Depression of Fungal Polygalacturonase Activity in Solanum

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Agricultural and Environmental Chemistry

Depression of Fungal Polygalacturonase Activity in Solanum lycopersicum Contributes to Antagonistic Yeast-Mediated Fruit Immunity to Botrytis Laifeng Lu, Lifeng Ji, Qingqing Ma, Mingguan Yang, Shuhua Li, Qiong Tang, Liping Qiao, Fengjuan Li, Qingbin Guo, and Changlu Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00031 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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Journal of Agricultural and Food Chemistry

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Depression of Fungal Polygalacturonase Activity in Solanum lycopersicum

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Contributes to Antagonistic Yeast-Mediated Fruit Immunity to Botrytis

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Laifeng Lu 1*, Lifeng Ji 1, Qingqing Ma 1, Mingguan Yang 1, Shuhua Li 1, Qiong Tang 2,

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Liping Qiao 1, Fengjuan, Li 1, Qingbin Guo 1, Changlu Wang 1**

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1

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Ministry of Education, College of Food Engineering and Biotechnology, Institute for New Rural

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Development, Tianjin University of Science and Technology, Tianjin 300457, P.R. China

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2

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Food Technology and Equipment, Key Laboratory for Agro-Products Postharvest Handling of Ministry

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State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Food Nutrition and Safety,

College of Biosystems Engineering and Food Science, National Engineering Laboratory of Intelligent

of Agriculture, Zhejiang University, Hangzhou 310058, China

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12

13

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*Corresponding author.

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Tel: +86-022-60912453; Fax: +86-022-60912453; E-mail: [email protected]

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**Corresponding author.

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Tel: +86-022-60601154; Fax: +86-022-60601154; E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT

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The acquisition of susceptibility to necrotrophy over the course of ripening is one of the

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critical factors limiting shelf life. In this study, phytopathology and molecular biology were

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employed to explore the roles of pectinase in fruit susceptibility and ripening. Solanum

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lycopersicum fruit softened dramatically from entirely green to 50% red, which was

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accompanied by a continuously high expressed SlPG2 gene. The necrotrophic fungus

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Botrytis cinerea further activated the expression of SlPGs and SlPMEs to accelerate cell

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wall disassembly, while most of the polygalacturonase inhibitor proteins encoding genes

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expression were postponed in ripe fruit following the pathogen attack. Pectin induced the

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antagonistic yeast to secrete pectinolytic enzymes to increase fruit resistance against grey

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mould. The activities of pathogenic pectinase of B. cinerea were correspondingly depressed

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in the pectin-inducible yeast enzyme elicited ripe fruit. These data suggest that pectinase is

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a molecular target for regulation of disease resistance during fruit ripening.

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Keywords: Solanum lycopersicum; immune response; Rhodosporidium paludigenum;

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pectinolytic enzymes; pectin methylesterases.

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Introduction

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Ripening is a well-coordinated terminal stage of development that involves genetically

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determined alterations in physiological, biochemical and structural features of fleshy fruit,

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and it provides the benefits of de-greening, accumulation of sugars, acids and volatiles, and

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cell turgor variation.1 During the ripening process, cell wall-modifying enzymes are

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produced, causing textural changes in the fruit to make them softer and more palatable to

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seed dispersing animals, while these cell wall changes, which were evolved by plants to

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restrict pathogen penetration and infection, dramatically increase plant susceptibility to

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pathogen attack.2 Disruption of the primary cell wall during the early stages of fruit

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softening and the excess dissolution of the pectin-rich middle lamella in the deteriorating

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overripe stages are the main contributors to limited shelf life, which result in large

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economic losses of perishable horticultural products.2,3

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Pectins make up 35% of the primary cell wall in dicotyledonous plants and

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non-graminaceous (non-grass) monocots, and they play a central role in controlling the

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mechanical strength and rheological properties of the wall.4 The structure of pectin

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undergoes changes during fruit ripening that include a de-esterification of the

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methylesterified α-1, 4-linked galacturonic acid backbone by pectin methylesterases

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(PMEs), whose activity is regulated by an endogenous PME inhibitor (PMEI). The

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de-methylesterified pectin becomes a target for pectin-degrading enzymes, such as

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polygalacturonases (PGs), and pectate lyase-like enzymes, including pectate lyases and

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pectin lyases, resulting in the depolymerization or shortening of the primary poly-

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galacturonic acid skeleton.5 Ripening apples, bananas, grapes, papayas, peaches, pears and 3



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tomatoes possess relatively high levels of both PG and PME activities, although PG activity

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in peaches is initially activated when the fruits are mid to fully ripe. 6 Mutants in PGs or

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PMEs do not affect the fruit ripening or softening processes, but they display unexpected

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phenotypes, including resistance to cracking and the acquisition of postharvest pathogens.6

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Secreted pectinolytic enzymes are able to cause cell wall decomposition and tissue

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maceration. These enzymes thereby act as essential virulence factors that convert cell wall

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polymers into appropriate nutritional substrates for the invading microorganism in several

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necrotrophic fungal and bacterial, such as Aspergillus niger,7 Botrytis cinerea,8,9 Fusarium

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oxysporum,10

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canipestris13 and Xylella fastidiosa,14 allowing them entry into plant cells. Pectinases were

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the first cell wall-degrading enzymes to be induced and produced when F. graminearum

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was grown in infected tissues.15 The most extensively studied pectinolytic enzymes from

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fungal necrotrophs are those of B. cinerea, in which there are 28 pectinolytic enzymes

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potentially involved in the degradation of pectin.16 Bcpg1, Bcpg2, Bcpme1 and Bcpme2 are

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required for full virulence, and their knock-out mutants displayed strong reductions in

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virulence on tomato leaves and fruit.8,9,17,18 These virulence factors secreted by necrotrophs

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were found to induce host plants to cooperate in disease development through the

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manipulation of host cell wall-degrading enzymes.19 Controlling the activities of

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pectinolytic enzymes is important for increasing shelf life and for necrotrophic pathogens to

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achieve successful infection.2,20

Penicillium

digitatum,11

Ralstonia

Solanacearum,12

Xanthomonas

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Recognition of extracellular effectors proteins via direct interactions with receptor-like

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proteins/kinases or cell wall fragments produced by hydrolytic enzymes may subsequently 4


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become signalling molecules alerting the plant of an impending infection.21 An early

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physiological response by the plant may minimize or end an attack by phytopathogenic

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organisms. Several fungal endo-PGs are directly recognized by the AtRLP42 protein, an

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Arabidopsis leucine-rich repeat receptor-like protein, also known as RESPONSIVENESS

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TO BOTRYTIS POLGALACTURONASES (RBPG1), which results in RBPG1 transgenic

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plants

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Oligogalacturonides (OGs) derived from pectic homogalacturonan also have specialized

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functions, beyond those of structural components, in the elicitation of phytoalexin

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accumulation, defence-related genes and reactive oxygen species.23,24 Transgenic plants

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expressing a plant polygalacturonase-inhibiting protein (PGIP)-fungal PG chimaera under

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the control of a pathogen-inducible promoter resulted in the in vivo production of OGs and

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plants that were more resistant to the phytopathogens, B. cinerea, Pectobacterium

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carotovorum, and Pseudomonas syringae. The controlled release of OGs upon infection

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provided a valuable tool to protect plants against infectious diseases.25

having

increased

resistance

to

Hyaloperonospora

aradidopsidis.22

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Plant-associated microorganisms fulfil important functions for plant growth and health.

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Plant beneficial microbes including Bacillus, Pseudomonas, Serratia, Stenotrophomonas,

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Streptomyces, Ampelomyces, Coniothyrium and Trichoderma are well-studied model

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organisms that have been used to demonstrate the influence of beneficial microbes on plant

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health.26 It is possible to develop microbial inoculants included in biofertilizers, plant

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strengtheners, phytostimulants, and biopesticides for use in agricultural biotechnology. The

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use of biocontrol agents as an alternative to synthetic chemical fungicides is becoming

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popular around the world. Several postharvest diseases can now be controlled by microbial 5



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antagonists.27,28 A better understanding of the mode of action of postharvest biocontrol

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agents is critical for the advancement and successful implementation of postharvest

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biocontrol technology.27 Wilson et al. (1993) reported on their strategy to utilize fruit

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wounds to screen for potential yeast antagonists against postharvest rot organisms from

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unidentified microbial populations on fruit surfaces.29 This strategy aided in the rapid

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selection of a number of potential antagonists for the control of postharvest diseases of fruit

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around the world. By contrast, limited attention has been focused on the relationship

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between biological control microbes and the plant cell wall that they inhabit, especially on

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the most abundant compounds, such as pectin. Here, the occurrence of pectinase-related

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genes in S. lycopersicum fruit was described in response to the necrotrophic pathogen, B.

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cinerea, and an excellent beneficial yeast, Rhodosporidium paludigenum. Three

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polysaccharides in the plant cell wall were selected to examine the biological control

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mechanisms of R. paludigenum against grey mould in tomatoes. Plant pathology

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experiments and enzymatic tests were used to demonstrate the effect of the interaction

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between secreted yeast proteins and fruit endo-pectinases on fruit-induced disease with B.

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cinerea. Finally, we demonstrate that regulation of S. lycopersicum pectinase activity is an

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essential element in the ability of these biological strains to protect fruit from infection.

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Materials and methods

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Fruit materials

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Expanding tomato (S. lycopersicum) fruits were harvested at the MG stage (full-size green

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fruit), the PK stage (50% pink- or red-coloured fruit) and the RR stage (ripe red-coloured

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fruit, approximately breaker + 6 days with full red colour) in a greenhouse at an 6


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experimental orchard at the Dongli Economic Development Area Station (Tianjin, China).

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Fruit samples were harvested at the same time for physical, pathological, biochemical and

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molecular evaluation from 100 randomly selected individual plants without injuries or

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infections. At least 12 fruits were harvested for each biological replicate. Fruits were

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surface-sterilized in a 0.1% (v/v) sodium hypochlorite aqueous solution for 2 min, rinsed

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thoroughly with tap water, and then air-dried at 20 °C prior to the experiment.

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Microbial culture

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The biological control strain, R. paludigenum,30 was incubated in 250 g L-1 tomato dextrose

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broth for 36 h at 28 °C on a rotary shaker (3.3 s-1) in 250 mL Erlenmeyer flasks. To activate

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inducible enzyme secretion, the polysaccharide pectin (Sigma, USA), cellulose (Sigma,

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USA), or xylan (Macklin, China) were added to the tomato dextrose broth at a 1%

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concentration prior to sterilization. Yeast cells were collected by centrifugation at 2810 g

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for 15 min and then washed and resuspended twice in sterile water. The final concentration

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of the cell suspension was adjusted to 1 × 108 cells mL-1. B. cinerea was cultured for 7 d on

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200 g L-1 potato dextrose agar (PDB) at 25 °C in the dark.30 The spore concentration was

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determined by microscopic counting and then adjusted to 5 × 104 spore mL−1. The spore

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suspension used for inoculations was freshly prepared to ensure good viability.

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Infection assays

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Wounds (2 mm in depth and 5 mm in diameter) were created on the S. lycopersicum fruit

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surface using a sterile borer as described previously. Thirty microlitres of the R.

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paludigenum cell suspension or proteins isolated from its fermentation broth by ammonium

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sulphate precipitation (90% saturation) were added into the surface wound. Mock 7



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inoculations were performed using sterile distilled water. To test the effect of the yeast or

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its secretome on the induction of disease resistance in tomato fruits, 20 μL of the B. cinerea

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spore suspension (5 × 104 spore mL-1) was inoculated into a second wound 48 h after

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inoculation, at a distance approximately 1/4 of the perimeter of the fruit away from the

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initial wound. The percentages of infected wounds and the average diameter of the lesions

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were measured 36 h and 48 h after storage at 90-95% relative humidity and 25 °C.

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Population growth of R. paludigenum on the fruit surface and ability to antagonize B.

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cinerea spore germination and growth

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Fruit surface wounds inoculated with R. paludigenum were collected at different time

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intervals (0, 24, 48, 72 and 96 h) after harvest by removing the peel tissue with a cork borer.

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The resulting cylinders of excised tissue (2 mm deep × 1 cm wide) from three fruits were

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placed in a mortar with 15 mL of sterile distilled water and ground with a pestle. To

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evaluate the antagonistic capability of R. paludigenum towards B. cinerea, conidial

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suspensions were mixed with freshly harvested R. paludigenum cells (1 × 107 cells mL-1) in

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PDB medium. The spore growth of B. cinerea was then evaluated by microscopy after

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incubation at 25 °C for 18 h in the dark.

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Determination of PG, PME and pectinase inhibitor activities

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For enzyme extraction, fruit surface wounds were treated with 30 μL of pectinolytic

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enzymes of R. paludigenum fermentation broth for 24 h at 25 °C. Five grams of fresh

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pericarps was homogenized with 10 mL of ice-cold 95% ethanol and kept for 10 min at 4

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°C. After centrifugation at 16128 g for 20 min at 4 °C, precipitates were re-suspended in 10

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mL of ice-cold 80% ethanol, kept for 10 min and then centrifuged as described above. The 8


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precipitates were further extracted with 5 mL of 50 mmol L−1 sodium acetate buffer (pH 5.5,

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containing 1.8 mol L−1 NaCl), kept for 20 min and then centrifuged as above. The

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supernatants were collected as the crude enzyme extract for determining PG and PME

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activities according to Rodoni et al. (2010) with a little modification.31

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PG activity was assayed by monitoring the release of reductive sugar from

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polygalacturonic acid (Aladdin, China) using the 3,5-dinitrosalicylic acid (DNS) method

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with D-galacturonic acid monohydrate (Aladdin, China) as the standard. Crude enzyme

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extracts (1.0 mL) were incubated with an equal volume of 10 g L−1 polygalacturonic acid in

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50 mmol L−1 sodium acetate buffer (pH 5.5) for 1 h at 37 °C. Mock reactions were prepared

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by boiling the enzyme extract for 5 min before incubation under the same condition. The

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released reductive sugar was determined at 540 nm using the DNS reagent after heating at

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100 °C for 5 min. The PG activity was calculated and expressed as μg D-galacturonic acid

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monohydrate per hour per gram of fresh fruit.

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PME activity was assayed by determining the production of pectic acid during the

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pectin demethylation process. Ten millilitres of 1% (w/v) pectin (Aladdin, China) in

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3 mmol L−1 potassium phosphate buffer (pH 7.5) was mixed with 10 mL of enzyme

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extracts for 3 min at 37 °C. The mixtures were then adjusted to pH 7.5 with 0.05 mol L-1

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NaOH using neutral red (Aladdin, China) as an indicator, and the released pectic acid was

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quantified by the volume of neutralized NaOH during the next 30 min at 37 °C. The results

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were expressed as mmol pectic acid per hour per gram of fresh fruit.

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The pathogenic pectinase, which was extracted from B. cinerea fermentation broth

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using the ammonium sulphate precipitation (90% saturation) method and cultured in 250 g 9



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L-1 tomato dextrose broth for 3 d at 28 °C, was used to assay the inhibitory ability of

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inducible pectinase inhibitor in fruit. The fruit pectinase inhibitor was extracted with 50

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mmol L−1 sodium acetate buffer (pH 6.0, containing 1.0 mol L−1 NaCl, 1.0% (w/v)

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polyvinylpyrrolidone, and 0.2% (w/v) sodium bisulfite), kept for 1 h and then centrifuged at

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16128 g for 20 min at 4 °C.32 PG activity was measured by the DNS method described as

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above. The inhibitory activity of the pectinase inhibitor was expressed as the percentage

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reduction in the number of reducing ends liberated by pectinases in the presence of

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extracted PGIP or PMEI in tomato surface wounds.

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Reverse transcription-PCR and qRT-PCR

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Total RNA was extracted from homogenized tissue frozen in liquid nitrogen using Trizol

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reagent (Invitrogen, USA), and 1 µg of RNA per 20 µL reaction was used to generate

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first-strand cDNA with the PrimeScript® RT reagent kit with gDNA eraser (TaKaRa,

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China) according to the manufacturer’s instructions.

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For reverse transcription-PCR analysis of loosened plant cell wall-related genes in the

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tomato fruit, gene-specific primers (Supplemental Table S1) at a concentration of 0.2 μM

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each with the equivalent of 50 ng reverse-transcribed RNA template per reaction were used

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to amplify the corresponding cDNA sequences by PCR under the following thermal cycling

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conditions: 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 45 s,

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using TB Green™ Premix Ex Taq™ II (TaKaRa, China) in a 50 µL reaction. The melt curve

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conditions were 95 °C for 15 s, 60 °C for 1 h and 95 °C for 15 s. The melt curves and

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no-template controls were examined to ensure against primer-dimer formation and

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contamination. Fluorescent signals were collected during the annealing and extension 10


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cycles. Cycle threshold (Ct) values were determined using the autothreshold function. The

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housekeeping control adaptor protein-2 mu-adaptin (CAC) was amplified in parallel on

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each plate for normalization,33 and all values were adjusted so that the controls at all time

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points were set to zero. The ΔΔCt was calculated by subtracting the ΔCt values of the

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controls from the ΔCt values of all other measurements at that time point. The relative

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mRNA expression was derived by the following formula: 2 [exp (−ΔΔCt)].

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Fruit firmness and pH measurements

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Pericarp firmness was measured on four separated but equidistant peeled sites on the

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equator of each fruit using a digital-display type penetrometer (GY-4; Aidebao, Zhejiang,

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China), the probe p/1 with diameter of 3.5 mm was used. Firmness was expressed as the

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maximum force (N) attained during penetration. The dynamic pH values of the

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fermentation both of R. paludigenum with three kinds of polysaccharides in tomato

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dextrose were determined by a pH meter (GB11165; Mettler Toledo, Switzerland) after 96

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h of inoculation at 28 °C.

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Statistical analyses

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Statistical analyses using one-way analysis of variance (ANOVA) and Duncan’s multiple

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range tests were performed using SPSS/PC ver. II.x (SPSS Inc. Chicago, IL, USA). Data

228

are presented as the means of three replicated samples ± the standard deviation. A P-value

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of < 0.05 indicated statistical signiﬁcance. Figures were produced using Origin 8.0

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(Microcal Software, Inc., Northampton, MA). BMKCloud (https: //international.

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biocloud.net) was used to generate heatmaps of gene expression.

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Results 11



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SlPG, SlPGIP, SlPME, and SlPMEI isoforms and SlPL8 are co-expressed during

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ripening

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The hardness of freshly harvested S. lycopersicum fruit at three ripening stages (the MG

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stage, PK stage and RR stage) was measured to evaluate the index of fruit softening as an

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indicator of cell wall decomposition (Figure 1A). The fruit softened along with the ripening

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process, and the most dramatic changes appeared from the MG stage to the PK stage. The

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maximum force attained during penetration of MG stage fruit was 30.52±3.26 N, which

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was twice as high as what was attained for the fruit in the two latter stages. We next studied

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the related regulation of the SlPG, SlPGIP, SlPME, and SlPMEI isoforms and SlPL8

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expression. These genes dominate pectin degradation in cell wall, and SlEXP1 and SlCEL2

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expression is responsible for the extension of the cell wall, leading to the enlargement of

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the plant cell that occurs during the fruit ripening process (Figure 1B). A marked decrease

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in SlPG and SlPG1 expression and an increase in SlPG2 and SlPL8 expression were

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concurrently observed in S. lycopersicum fruit, along with fruit softening during the

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transition from the MG to the PK stage. In PK stage fruit, expression of the SlPG2 and

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SlPL8 genes (two genes that are supposedly responsible for pectin depolymerization and

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solubilization in the cell wall) was increased by more than 11.0- and 1.1-fold (Log base 2),

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respectively, compared to that in the MG stage. The expression of SlCEL2 was also found

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to be up regulated starting during the MG stage and ending in the PK stage, after which its

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expression trended downward. Expression of the SlPME isoforms decreased after the MG

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stage and displayed a secondary increase during the PK stage (Figure 1B). A high SlPME

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isoform mRNA content was supposed to accumulate before both the MG and RR stages 12


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and to continuously transcribe into pectinolytic enzymes during ripening.

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SlPGIPs and SlPMEs differentially respond to B. cinerea during ripening

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To study whether pectin modification-related gene, including the SlPGIPs and SlPMEIs,

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are required for host immunity against B. cinerea infection, the relative expression levels of

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19 genes that could be classified into 7 isoforms, including SlPG, SlPGIP, SlPME, SlPMEI,

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SlPL, SlEXP and SlCEL was measured during B. cinerea infection at different time

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intervals during the MG and RR maturation stages. The necrotrophic fungus B. cinerea has

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the ability to significantly alter the expression of polygalacturonase-, pectin lyase-,

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cellulase-, and expansin-encoding genes, as well as the expression of their inhibitors,

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during both the MG (Figure 2A) and RR stages (Figure 2B). The genes SlPG, SlPL8,

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SlEXP1 and SlCEL2 were observed to be up regulated by treatment with the pathogen B.

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cinerea during the initial 6 hours of infection in both the MG and RR stages, whereas the

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polygalacturonase-encoding gene SlPG1 was only significantly up regulated by treatment

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with B. cinerea in MG stage fruit (Figure 2). Significantly reduced expression of SlPG,

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SlPL8, SlEXP1 and SlCEL2 appeared in the period following B. cinerea inoculation of fruit

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in the RR stage (Figure 2 B). Additionally, B. cinerea infection markedly reduced the

271

expression of the major maturity-related SlPG2 genes at initial time of infection in MG

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stage fruit, while no B. cinerea-induced SlPG2 expression was detected in RR stage (Figure

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3). These data suggested that B. cinerea mediated a temporal and intense differential

274

response to pectinase-related gene expression in the host fruit during ripening.

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Next, the effect of B. cinerea infection on the gene expression of host pectin

276

methylesterase was studied, including SlPME1 and SlPME2, during fruit ripening (Figure 13



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2). We observed a decrease in expression of both SlPME1 and SlPME2 in the mechanically

278

wounded fruits treated with water at both fruit stages, and to a certain extent, B. cinerea

279

treatment alleviated the reduced expression of SlPME1 in MG stage fruit and SlPME2 in

280

the RR stage (Figure 3). The reduced expression of SlPME2 was automatically recovered

281

and reversed by the necrotrophic pathogen in RR stage fruit during the final stage (36 h) of

282

testing (Figure 3B). No consistent changes were found for SlPME1, SlPME2, SlPME3 or

283

SlPME31 expression after attack by B. cinerea during the two ripening stages (Figure 2).

284

To test whether pectinase inhibitors are involved in host immune responses to B.

285

cinerea infection, we analysed the expression of three polygalacturonase inhibitors, four

286

pectin methylesterase inhibitors and the SlPPE8B gene in harvested MG and RR stage fruit.

287

The necrotrophic fungus B. cinerea indeed has the ability to significantly up regulate the

288

expression of SlPGIP80.4, SlPPE8B, SlPMEI61 and SlPMEI51 during the initial 6 hours

289

after pathogen inoculation in MG stage fruit, but not in RR stage fruit. In terms of the

290

response time, the activation of fruit pectinase inhibitors appeared to be delayed for 6 to 12

291

h in the RR stage compared to the MG stage following pathogen attack, with the exception

292

of the PG inhibitor PGIP26.1 (Figure 2B). In MG stage fruit, after the initial 6 h elicitation

293

period, B. cinerea induced reduced SlPGIP80.4 expression during the next 30 hours, and

294

this reduction in expression was not observed in RR stage fruit (Figure 3A, 3B). Higher

295

expression of SlPMEI51 elicited by the B. cinerea pathogen was detected at the beginning

296

of infection, but this increased expression was suppressed in the remainder of the RR stage

297

probably leading up to disease symptom appearance.

298

Polysaccharides of plant cell walls enhanced the biological control function of yeast 14


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To study the interaction between plant beneficial microbes and polysaccharides of the plant

300

cell wall, pectin, cellulose and xylan were co-cultured with the yeast R. paludigenum in

301

tomato medium, and their ability to control B. cinerea infection was subsequently tested

302

(Figure 4A, 4B). As expected, the yeast R. paludigenum (1×107 cells mL-1) significantly

303

reduced the occurrence of grey mould disease and symptom development in tomato fruits

304

and could totally prevent the invasion of B. cinerea infection when the number of mould

305

spores was lower than 1×104 spores mL-1 in a wound. Moreover, we observed that ability of

306

the yeast R. paludigenum to control B. cinerea infection was markedly enhanced by three

307

polysaccharides of the cell wall. Among the polysaccharides, pectin and cellulose had the

308

best performance in preventing disease in fruit. At 36 h after pathogen inoculation, the

309

percentages of infection in the pectin co-cultured R. paludigenum (PERP) treatment and

310

cellulose co-cultured R. paludigenum (CERP) treatment groups were 12.5% and 21.3%,

311

respectively, which was a 58.3% and 49.5% reduction, respectively, compared to the

312

control tomato medium cultured R. paludigenum (TMRP) treatment (Figure 4A). The cell

313

wall polysaccharides helped R. paludigenum to protect the mechanical wounds of the fruit

314

and to prevent the wounds from being infected by necrotrophic fungi, such as B. cinerea.

315

Next, we explored the mechanism behind the increased ability of R. paludigenum to

316

control grey mould infection by monitoring the population dynamics of this yeast on fruit

317

surface wounds as an indicator of its ability to colonize the fruit surface (Figure 4C), as

318

well as its ability to antagonize B. cinerea spore germination and growth (Figure 4E, 4F). A

319

significant increase in the population of R. paludigenum was detected on the PERP- and

320

CERP-treated surface wounds at 24 h and 72 h after inoculation at 28 °C. By contrast, 15



Page 16 of 46

321

during the storage period, no significant changes in the yeast population were found in

322

xylan co-cultured R. paludigenum (XYRP) treated surface wounds compared with that for

323

the TMRP treatment (Figure 4C). Similarly, we observed that the pectin and cellulose

324

co-cultured R. paludigenum significantly inhibited the spore germination of B. cinerea in

325

PDB medium compared to the TMRP-treated and control-treated groups. CERP treatment

326

displayed a higher antagonism of B. cinerea spore germ tube elongation, with a 59.5%

327

reduction compared to the TMRP groups (Figure 4F). The increased antagonism of R.

328

paludigenum was one of the major contributors to the increased ability of CERP and PERP

329

to control B. cinerea infection.

330

It is worth noting that the polysaccharides of cell wall in sterilized tomato medium

331

culture were decomposed and utilized by R. paludigenum during the co-culture process.

332

Pectinolytic enzyme was supposed to exist in the secretome of the biological control agent

333

R. paludigenum to partially degrade pectin in fruit to play an important role in its survival

334

on fruit surface. We next detected the release of polysaccharide residues with free protons

335

(Figure 4D). The amount of available hydrogen was increased in CERP and PERP culture

336

media, which suggested that small molecular units, such as galactosidonic acid or

337

oligogalacturonides, may have been generated in the media through the enzymatic

338

degradation of PME and PG when they were highly inducible expressed and present.

339

R. paludigenum inducible secreted proteins aided in disease control

340

We next wanted to further explore whether the improvements in the ability of R.

341

paludigenum to control B. cinerea after co-culture with different plant cell wall

342

polysaccharides were due to the recognition of these enzymes as microbe-associated 16


Page 17 of 46


343

molecular patterns (MAMPs) or to the recognition of the polysaccharide breakdown

344

products as damage-associated molecular patterns (DAMPs). Thus, the polysaccharide-

345

inducible secreted proteins of R. paludigenum and the corresponding fermentation

346

supernatants were extracted and collected, respectively, from TMRP, PERP, CERP and

347

XYRP fermentation broth at 48 h after inoculation with a seed solution at 28 °C. We

348

observed that the polysaccharide-inducible secreted proteins from PERP, CERP and XYRP

349

fermentation broth induced a visible reduction in B. cinerea disease incidence of S.

350

lycopersicum fruit, with the exception of TMRP, which was co-cultured with no additional

351

polysaccharide (Figure 5A). In addition, the polysaccharide-inducible secreted proteins of

352

PERP decreased the lesion diameter of B. cinerea infections (Figure 5B) and showed a

353

better ability to activate the host immune system and/or a higher efficacy in producing

354

polysaccharide breakdown products. Additionally, we measured the ability of fermentation

355

supernatants from TMRP, PERP, CERP and XYRP fermentation broth to control B.

356

cinerea infection in tomatoes. None of the supernatants provided a beneficial effect on

357

protecting fruit from pathogen invasion (Figures 5C, 5D), suggesting that the

358

polysaccharide breakdown products were sequestered by R. paludigenum during

359

fermentation or that the polysaccharide concentrations are too low in the supernatants to

360

induce host resistance.

361

Secreted proteins from PERP fermentation broth altered host pectinase activities

362

Previous results demonstrated that tomatoes could regulate their pectinase-related gene

363

expression and immune response to B. cinerea during both stages of ripening. Therefore,

364

we hypothesized that PERP secreted protein (PERP-P) treatment could regulate host 17



Page 18 of 46

365

pectinase-related gene expression and activity in a similar way, such as by decreasing the

366

expression of the SlPGs and SlPMEs genes or by increasing the expression of SlPGIPs and

367

SlPMEIs inhibitors in S. lycopersicum fruit as a means of inducing resistance to B. cinerea

368

infection. During both the MG and RR stages, treatment with PERP-P for 24 h led to a

369

marked reduction in SlPG expression and induced higher expression of SlPGIP80.4,

370

SlPGIP26.1 and SlPGIL97.3 compared to the water-treated S. lycopersicum (WTSL)

371

control group (Figure 6A). Of note, two pectin methylesterases, SlPME1 and SlPME2, were

372

also down regulated by the PERP-P treatment compared to the control treatment. Increases

373

in the expression of the other three pectin methylesterase-encoding genes, SlPME3,

374

SlPME31 and SlPMEU1, and two PME inhibitor-encoding genes, SlPMEI51 and PMEI61,

375

were observed during both the MG and RR stages (Figure 6A), which suggested that the

376

pectin methylesterase inhibitors were involved in the induction of host disease resistance by

377

yeast. Furthermore, we detected an increase in the expression of the genes SlEXP1 and

378

SlPL8 during the MG and RR stages. By contrast, SlCEL2 expression was unchanged in the

379

PERP-treated fruit during the MG stage, but it decreased during the RR stage compared to

380

the WTSL control group (Figure 6A). When we changed the gene expression internal

381

reference from the water control to the TMRP-P treated S. lycopersicum (YSTL), we found

382

that the expression of the SlPGs, SlPME1 and SlPME2 were reduced, while the SlPGIPs

383

and SlPMEI mRNA content was increased to enhance the disease resistance in fruit by

384

PERP-P (Figure 6B). A marked reduction in SlPL8 and SlCEL expression was also detected

385

at the same time in RR stage fruit, indicating a decrease in cell wall decomposition

386

occurred after PERP-P treatment. 18


Page 19 of 46


387

To further verify our hypothesis, we monitored the activities of PG and PME in

388

PERP-P-treated fruit wound tissue. An increase in PG activity in PERP-treated tissue was

389

observed during both the MG and RR stages. Additionally, the PG activity in PERP-treated

390

RR stage fruit was much higher than in the other stages, which was consistent with the up

391

regulation of SlPG1 expression and the alleviation of SlPG2 repression (Figure 6C).

392

Moreover, we detected a decrease in PME activity in PERP-treated RR stage fruit, but not

393

in MG stage fruit, and no significant changes were observed in the PME activity of

394

PERP-treated MG stage fruit, confirming the previous results that PERP-P-treatment down

395

regulated SlPME1 and SlPME2 expression in tomatoes (Figure 6D).

396

Pectinases are virulence factors secreted by necrotrophic pathogens, such as B. cinerea,

397

to overcome the obstacle of the tomato fruit cell wall. The beneficial yeast R. paludigenum

398

induces host cooperation in disease resistance through the manipulation of host cell wall

399

degrading enzymes. We next wanted to understand what would happen to the virulence

400

factors activity after the host interacted with R. paludigenum. We assayed the inhibitory

401

activity of PERP-P-induced pectinase inhibitors against pectinases secreted by B. cinerea in

402

fruit surface wounds by mixing them together (Table 1). The results showed that PERP-P

403

induced pectinase inhibitors in RR stage fruit depressed the release of galactosidonic acid

404

by B. cinerea pectinase pectin hydrolysis. By contrast, PERP-P induced pectinase inhibitors

405

in MG stage fruit were completely ineffective or not needed. This finding was positively

406

associated with the changes in PME activity demonstrated in Figure 6D. Thus,

407

PERP-P-mediated PME reduction and increases in PGIP and PMEI activities were

408

supposed to play an important role in regulation of ripe fruit defence response to B. cinerea. 19



Page 20 of 46

409

Discussion

410

The acquisition of B. cinerea susceptibility is a hallmark of tomato fruit ripening; ripe

411

fleshy fruit are more susceptible to disease and decomposition than unripe green fruit.34,35

412

Here, we showed that the co-expression of a number of genes, including SlPGs, SlPMEs,

413

SlCEL2, SlEXP1 and SlPL8, likely leads to a dramatic reduction in fruit hardness during

414

ripening. To accelerate the maturation process, the necrotrophic fungus B. cinerea has the

415

ability

416

expansin-encoding gene expression during both the MG and RR stages. Gene expression of

417

pectinase-related inhibitors, a major part of the fruit immune system, appears to be

418

postponed for 6 to 12 hours in RR stage tomatoes following pathogen attack, a

419

phenomenon that coincides with the degradation of fruit disease resistance during ripening.

420

Considering that Bcpg1 B cinerea mutants have significantly reduced virulence on tomato

421

leaves and fruits, as well as on apple fruits, which markedly limits the growth of the lesion

422

beyond the inoculation spot,8 we initially considered the possibility that pre-activating fruit

423

PGIP and PMEI activity by inducible pectinase from the beneficial microbe R.

424

paludigenum may be helpful in reducing losses caused by necrotrophic B. cinerea infection

425

in tomatoes. As expected, two plant cell wall polysaccharides, including pectin and

426

cellulose, could be utilized by R. paludigenum, which enhanced its ability to prevent

427

postharvest disease and increased its ability to colonize the fruit surface where the

428

antagonistic yeast was selected from. Secreted proteins from PERP induced a visible

429

reduction in the disease incidence of grey mould across the entire tomato fruit. PERP

430

provides a stable, convenient and continuous MAMP or DAMP supply to ripening

to

significantly

alter

polygalacturonase-,

pectin

lyase-,

cellulase-,

and

20


Page 21 of 46


431

horticultural products and avoids the complexity and high cost in fabrication, which has

432

previously limited the popularization of MAMPs/DAMPs.

433

The plant cell wall is the primary interface where most plant-microbe interactions

434

occur and the main physical and molecular line of defence evolved by plants to restrict

435

pathogen penetration and the spread of infection.20 Ripening changes the structure and

436

composition of the cell wall, altering the “battle path” between horticultural products and

437

microorganisms. Pectin disassembly is particularly extensive and is associated with the

438

later stages of ripening, as well as fruit deterioration in the overripe stages.36 We found that

439

expression of the genes SlPG2, SlPL8 and SlCEL2 was concurrently increased, with the

440

most dramatic changes appearing from the MG stage to the PK stage. Additionally, SlPG2

441

expression was more than 11.0-fold higher (Log base 2) in the PK stage than in the MG

442

stage, indicating that pectin and cellulose are depolymerized and solubilized in the S.

443

lycopersicum fruit cell wall during fruit ripening. After the PK stage, the expression of the

444

SlPG2 gene continued to increase, whereas SlPL8 and SlCEL2 mRNA expression trended

445

downward. This observation was consistent with previous theories that initiation of the

446

degradation of the ripening cell wall and the early stages of fruit softening are accompanied

447

by a decrease in the molecular size of hemicellulose. Pectin disassembly was previously

448

found to be responsible for the deterioration that occurs in the overripe stages.36 While

449

PG-dependent pectin disassembly has been extensively studied in ripening tomatoes for

450

more than 30 years, it was concluded that it is not necessary for normal ripening and

451

softening. In soft fruits, such as tomatoes, the dissolution of the pectin-rich middle lamella

452

begins early in ripening.37 Suppression of PG activity only slightly reduces fruit softening, 21



Page 22 of 46

453

but it extends the shelf life of fruit.6 The cell wall-modifying proteins LePG and LeExp1

454

have been shown to facilitate susceptibility and were induced only in susceptible

455

RIPENING INHIBITOR fruits but not in resistant NON-RIPENING fruits.35 In line with

456

these observations, we hypothesized that the increased SlPG2, SlPL8 and SlEXP1

457

expression may contribute to ripening-related disassembly of the cell wall and the increased

458

susceptibility to B. cinerea.

459

Phytopathogenic fungi usually secrete pathogen-derived molecules as a strategy to

460

successfully invade and infect plant tissues but also utilize host-derived molecules that

461

favour the pathogenicity process. To overcome the Arabidopsis cell wall barrier,

462

necrotrophic fungi such as B. cinerea and P. carotovorum force the host to rapidly express

463

AtPME3 to act as a susceptibility factor and work in concert with their own arsenal of

464

hydrolytic enzymes for the initial colonization of the host tissue.38 Accordingly, our data

465

show that B. cinerea treatment up regulated SlPG, SlPL8, SlEXP1 and SlCEL2 expression

466

during the initial 6 hours of infection in both the MG and RR stage fruits, indicating that B.

467

cinerea could be utilizing tomato-derived cellulase and pectinolytic enzymes to accelerate

468

cell wall decomposition. This increased expression was found to be more consistent in RR

469

stage fruit; furthermore, SlPME2, SlPS2 and SlPG2 had higher levels of expression in

470

response to grey mould in RR stage fruit compared to the levels in MG stage fruit,

471

suggesting that ripe fruit may favour the pathogenic process of B. cinerea and that the

472

pathogen itself may initiate the induction of susceptibility through its sensing of fruit

473

ripening status. Regulators of fruit ripening, such as NOR, participate in regulating

474

ripening-associated pathogen susceptibility, such that mutants of SlPG and SlExp1 cannot 22


Page 23 of 46


475

be induced by B. cinerea.3 These data implied that PGs are one of the most important

476

connections between increased B. cinerea susceptibility and fruit ripening.

477

Plants have evolved different PGIPs and PMEIs to regulate processes related to plant

478

growth and specific recognition of virulence factors, including pectolytic enzymes

479

produced by fungi that inhibit damage caused by them.39,40 Transgenic tomato plants

480

expressing the pear fruit pPGIP, which was detected in the cell wall protein fractions of all

481

transgenic tissues, reduced the growth of B. cinerea on ripe tomato fruit and diminished

482

tissue breakdown by as much as 15%.41 Our results from treatments with B. cinerea at two

483

ripening stages showed that a number of SlPGIPs and SlPMEIs have enhanced expression,

484

indicating that the tomato fruit can employ pectolytic enzyme inhibitors to depress external

485

PG or to inhibit internal pectin de-methylesterification to respond to B. cinerea invasion.

486

Among those inhibitor-encoding genes, SlPGIP80.4, SlPPE8B, SlPMEI61 and SlPMEI51

487

were rapidly and specifically activated by the B. cinerea necrotrophic fungus during the

488

MG stage. With the exception of the PG inhibitor PGIP26.1, these genes displayed a much

489

slower and weaker response during the RR stage. Considering the fact that pectin

490

methylesterase activity is required for susceptibility to B. cinerea in Arabidopsis (although

491

it does not inhibit fungal PMEs),20,42,43 the delay in PGIP and PMEI expression indicates

492

that the fruit immunity response was postponed in the RR stage following pathogen attack.

493

This effect coincides with the degradation of fruit disease resistance of ripening.

494

Furthermore, by restricting the action of PGs secreted by pathogens, PGIPs in the host not

495

only hinder pectin degradation but also favour the accumulation of elicitor-active OGs

496

fragments.44,45 Transgenic Arabidopsis plants expressing a fungal PG and plant PGIP gene 23



Page 24 of 46

497

chimaera under the control of a pathogen-inducible promoter are more resistant to

498

phytopathogens.25 Unfortunately, elevated expression levels of the chimaera caused the

499

accumulation of salicylic acid, reduced plant growth, restricted the population of

500

over-expressed PGIP genes, caused the accumulation of OGs in transgenic plants, and

501

eventually led to plant death.

502

Fruit surfaces injuries inflicted during harvest and subsequent handling represent an

503

ideal site of infection for necrotrophic pathogens and are considered to be a major cause of

504

decay in perishable horticultural products.28 The wound-invading necrotrophic fungus, B.

505

cinerea, which causes postharvest grey mould on apples, tomatoes, and pears among other

506

fruits, requires nutrients for germination and initiation of the pathogenic process.

507

Antagonistic species could protect fruit wounds from necrotrophic pathogens by direct

508

application to the targeted area (fruit wounds) using existing delivery systems (drenches,

509

line sprayers, on-line dips).46 We previously showed that R. paludigenum Fell & Tallman, a

510

potential biocontrol yeast, can significantly reduce decay through a wide range of functions,

511

including space and nutrient competition,30 detoxification of mycotoxins47 and by inducing

512

resistance. In the present study, we also observed a significant reduction in the occurrence

513

of grey mould and the development of disease symptoms in tomatoes. We have long

514

overlooked the fact that all antagonistic yeasts can grow rapidly in surface wounds of fruits

515

and that the yeast have the potential ability to decompose the polysaccharides of the fruit

516

cell wall and compete for nutrition and space with pathogens. By pre-co-culturing yeast

517

with polysaccharides from the fruit cell wall, we can restore the degradation activities of

518

these organisms in the laboratory and enhance their environmental adaptability and 24


Page 25 of 46


519

biological control abilities. As expected, among the three plant cell wall polysaccharides

520

tested, pectin and cellulose had the ability to enhance the ability of antagonistic yeast to

521

control B. cinerea infection in tomatoes. This enhanced ability to control infection was

522

partially contributed to by the rapid growth of the yeast within the surface wounds of the

523

fruit and its antagonistic effect on the spore germination of B. cinerea. More importantly,

524

PERP-P and CERP-P induced disease resistance in whole fruit, indicating that the yeast

525

possessed inducible polysaccharide hydrolase-encoding genes in their genome, which

526

favour their ability to adapt quickly to changes or stresses in the external environment.

527

Damaged fruit tissue can release DAMPs, such as oligogalacturonides and cellobiose,

528

which can be recognized by plant cell receptors to trigger downstream host defence

529

mechanisms.48,49 Yeast antagonists have the ability to interact with host tissues, particularly

530

with the wounds, to increase the cicatrization process, which raises the assumption that

531

yeast cells can induce resistance processes in the fruit skin through elicitors that are either

532

secreted or component of their cell walls.46 After incubating secreted proteins from PERP,

533

CERP and XYRP with plant wounds for 48 h, we detected a systematic disease resistance

534

in new adjacent wounds of PERP-P-treated groups. This result indicated that the pectinase

535

or polysaccharide breakdown products, such as OGs, may be recognized by the leucine-rich

536

repeat receptor-like protein, RBPG1, eventually resulting in accelerated wound healing

537

processes that serve as a means of protection against pathogen invasion. Orozco-Cardenas

538

et al. (2001) reported that PG mRNA is systemically induced by wounds in tomato leaves

539

and that PG activity is induced by systemin and methyl jasmonate.50 PERP-P induced a

540

much higher SlPGs expression and PGIP activity in B. cinerea-infected tomatoes during the 25



Page 26 of 46

541

RR stage compared to wounding alone, this finding and earlier observations suggest that

542

external PGs, which act as a MAMP to warn fruit of danger, could accelerate the hydrolysis

543

of cell wall polysaccharides and production of MAMP in whole fruit.50 So far, limited

544

information of fruit cell wall-associated defence, which operates via the inhibition of fungal

545

cell wall-degrading enzymes, has been reported. The present study initially considered the

546

possibility that pre-activating fruit PGIP and PMEI activity by inducible secreted pectinases

547

in PERP could reduce the losses caused by necrotrophic B. cinerea infection of tomatoes.

548

We found that the inducible yeast polysaccharide hydrolases of PERP-P, CERP-P and

549

XYRP-P activated the host immune response to B. cinerea in S. lycopersicum during RR

550

stage but that the supernatants of TMRP-S, PERP-S, CERP-S and XYRP-S fermentation

551

broth did not. Using reverse transcription-PCR and qRT-PCR, we found that SlPGIPs and

552

SlPMEIs were up regulated and SlPGs, SlPME1, and SlPME2 were down regulated in the

553

PERP-P treatment groups compared to the TMRP-P groups. Notably, the PERP-P treatment

554

also appeared to accelerate the fruit maturation process by increasing the expression of

555

SlPG2 and SlEXP1 in RR stage fruit. Here we initially revealed the use of fruit cell wall

556

polysaccharides by yeast and then showed that released DAMPs, including pectolytic

557

enzymes or elicitor-active OGs fragments, induce resistance in tomatoes and play an

558

important role in the ability of the yeast R. paludigenum to control infection. On the basis

559

of the present findings, we probably found a convenient and continuous MAMP/DAMP

560

bio-supply for use in reducing ripening-associated decay of fresh horticultural products.

26


Page 27 of 46


562

FUNDING SOURCES

563

This work was supported by the National Natural Science Foundation of China [grant

564

numbers

31701668,

565

numbers

16YFZCNC00700];

566

17JCQNJC14300]; Open Project Program of State Key Laboratory of Food Nutrition and

567

Safety, Tianjin University of Science & Technology [No. SKLFNS-KF-201827]; The

568

Project program of Key Laboratory of Food Nutrition and Safety, Ministry of Education

569

[No. 2018011]; The Foundation of Tianjin University of Science and Technology, Institute

570

for New Rural Development [No. xnc201706], P. R. China.

31571897];

Key Technologies R & D Program of Tianjin

Natural Science Foundation of Tianjin

[grant

[grant

numbers

571 572

CONFLICT OF INTEREST

573

The authors declare that they have no conflict of interest.

27



Page 28 of 46

575

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auxin-regulated rolB gene expression in tobacco leaf explants. Plant Physiol. 2000, 122,

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1379-85.

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(24)Denoux, C.; Galletti, R.; Mammarella, N.; Gopalan, S.; Werck, D.; De Lorenzo, G.;

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Ferrari, S.; Ausubel, F. M.; Dewdney, J., Activation of defense response pathways by OGs

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and Flg22 elicitors in Arabidopsis seedlings. Mol. Plant 2008, 1, 423-445.

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(25)Benedetti, M.; Pontiggia, D.; Raggi, S.; Cheng, Z. Y.; Scaloni, F.; Ferrari, S.; Ausubel,

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F. M.; Cervone, F.; De Lorenzo, G., Plant immunity triggered by engineered in vivo release

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of oligogalacturonides, damage-associated molecular patterns. Proc. Natl. Acad. Sci. U. S.

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A. 2015, 112, 5533-5538.

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(26)Berg, G., Plant–microbe interactions promoting plant growth and health: perspectives

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for controlled use of microorganisms in agriculture. Appl. Microbiol. Biotechnol. 2009, 84,

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11-18.

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(27)Droby, S.; Wisniewski, M.; Macarisin, D.; Wilson, C., Twenty years of postharvest

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biocontrol research: Is it time for a new paradigm? Postharvest Biol. Technol. 2009, 52,

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137-145.

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(28)Janisiewicz, W. J.; Korsten, L., Biological control of postharvest diseases of fruits.

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Annu. Rev. Phytopathol. 2002, 40, 411-441.

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(29)Wilson, C. L.; Wisniewski, M. E.; Droby, S.; Chalutz, E., A selection strategy for

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microbial antagonists to control postharvest diseases of fruits and vegetables. Sci. Hortic.

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(Amsterdam, Neth.) 1993, 53, 183-189. 31



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(30)Wang, Y. F.; Bao, Y. H.; Shen, D. H.; Feng, W.; Yu, T.; Zhang, J.; Zheng, X. D.,

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Biocontrol of Alternaria alternata on cherry tomato fruit by use of marine yeast

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Rhodosporidium paludigenum Fell & Tallman. Int. J. Food Microbiol. 2008, 123, 234-239.

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(31) Rodoni, L.; Casadei, N.; Concellón, A.; Chaves Alicia, A. R.; Vicente, A. R., Effect of

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short-term ozone treatments on tomato (Solanum lycopersicum L.) fruit quality and cell

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wall degradation. J. Agr. Food Chem. 2010, 58, 594-599.

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(32) Faize, M.; Sugiyama, T.; Faize, L.; Ishii, H., Polygalacturonase-inhibiting protein

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(PGIP) from Japanese pear: possible involvement in resistance against scab. Physiol. Mol.

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Plant Pathol. 2003, 63, 319-327.

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(33)Exposito-Rodriguez, M.; Borges, A. A.; Borges-Perez, A.; Perez, J. A., Selection of

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internal control genes for quantitative real-time RT-PCR studies during tomato

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development process. BMC Plant Biol. 2008, 8, 12.

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Blanco-Ulate, B.; Vincenti, E.; Cantu, D.; Powell, A. L. T., Ripening of tomato

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fruit and susceptibility to Botrytis cinerea. In Botrytis – the fungus, the pathogen and its

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management in agricultural systems, Fillinger, S.; Elad, Y., Eds. Springer International

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Publishing: Cham, 2016; pp 387-412.

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(35)Cantu, D.; Blanco-Ulate, B.; Yang, L.; Labavitch, J. M.; Bennett, A. B.; Powell, A. L.

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T., Ripening-regulated susceptibility of tomato fruit to Botrytis cinerea requires NOR but

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not RIN or Ethylene. Plant Physiol. 2009, 150, 1434-1449.

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(36)Pan, X.; Zhu, B.; Zhu, H.; Chen, Y.; Tian, H.; Luo, Y.; Fu, D., iTRAQ protein profile

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analysis of tomato green-ripe mutant reveals new aspects critical for fruit ripening. J.

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(37)Crookes, P. R.; Grierson, D., Ultrastructure of tomato fruit ripening and the role of

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polygalacturonase isoenzymes in cell wall degradation. Plant Physiol. 1983, 72, 1088-93.

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(38)Raiola, A.; Lionetti, V.; Elmaghraby, I.; Immerzeel, P.; Mellerowicz, E. J.; Salvi, G.;

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Cervone, F.; Bellincampi, D., Pectin methylesterase is induced in Arabidopsis upon

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infection and is necessary for a successful colonization by necrotrophic pathogens. Mol.

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Plant-Microbe Interact. 2010, 24, 432-440.

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(39)De Lorenzo, G.; D'Ovidio, R.; Cervone, F., The role of polygalacturonase-inhibiting

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proteins (PGIPS) in defense against pathogenic fungi. Annu. Rev. Phytopathol. 2001, 39,

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313-335.

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(40)Bonavita, A.; Carratore, V.; Ciardiello, M. A.; Giovane, A.; Servillo, L.; D’Avino, R.,

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Influence of pH on the structure and function of Kiwi pectin methylesterase inhibitor. J.

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Agric. Food Chem. 2016, 64, 5866-5876.

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(41)Powell, A. L. T.; van Kan, J.; ten Have, A.; Visser, J.; Greve, L. C.; Bennett, A. B.;

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Labavitch, J. M., Transgenic expression of pear PGIP in tomato limits fungal colonization.

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Mol. Plant-Microbe Interact. 2000, 13, 942-950.

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(42)Giovane, A.; Servillo, L.; Balestrieri, C.; Raiola, A.; D'Avino, R.; Tamburrini, M.;

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Ciardiello, M. A.; Camardella, L., Pectin methylesterase inhibitor. Biochim. Biophys. Acta,

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Proteins Proteomics 2004, 1696, 245-252.

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(43)Kubicek, C. P.; Starr, T. L.; Glass, N. L., Plant cell wall-degrading enzymes and their

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secretion in plant-pathogenic fungi. Annu. Rev. Phytopathol. 2014, 52, 427-51.

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(44)Ferrari, S.; Savatin, D. V.; Sicilia, F.; Gramegna, G.; Cervone, F.; Lorenzo, G. D.,

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Oligogalacturonides: plant damage-associated molecular patterns and regulators of growth 33



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and development. Front Plant Sci. 2013, 4, 49.

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(45)Huckelhoven, R., Cell wall - Associated mechanisms of disease resistance and

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susceptibility. In Annu. Rev. Phytopathol., Annual Reviews: Palo Alto, 2007; Vol. 45, pp

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101-127.

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(46)Spadaro, D.; Droby, S., Development of biocontrol products for postharvest diseases of

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fruit: The importance of elucidating the mechanisms of action of yeast antagonists. Trends

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Food Sci. Technol. 2016, 47, 39-49.

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(47)Zhu, R. Y.; Feussner, K.; Wu, T.; Yan, F. J.; Karlovsky, P.; Zheng, X. D.,

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Detoxification of mycotoxin patulin by the yeast Rhodosporidium paludigenum. Food

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Chem. 2015, 179, 1-5.

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(48)Robert-Seilaniantz, A.; Grant, M.; Jones, J. D., Hormone crosstalk in plant disease and

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defense: more than just jasmonate-salicylate antagonism. Annu. Rev. Phytopathol. 2011, 49,

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317-43.

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(49)Souza, C. A.; Li, S.; Lin, A. Z.; Boutrot, F.; Grossmann, G.; Zipfel, C.; Somerville, S.

721

C., Cellulose-derived oligomers act as damage-associated molecular patterns and trigger

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defense-like responses. Plant Physiol. 2017, 173, 2383-2398.

723

(50)Orozco-Cardenas, M. L.; Narvaez-Vasquez, J.; Ryan, C. A., Hydrogen peroxide acts as

724

a second messenger for the induction of defense genes in tomato plants in response to

725

wounding, systemin, and methyl jasmonate. Plant cell 2001, 13, 179-191.

34


Page 35 of 46


727

FIGURE CAPTIONS

728

Figure 1. Expression patterns of cell wall-modifying enzyme encoding genes during tomato

729

fruit ripening. A, Hardness of tomato fruit during MG stage (full-size green fruit), PK stage

730

(50% pink- or red-coloured fruit) and RR stage (ripe red-coloured fruit, approximately

731

breaker + 6 days with full red colour). B, Relative expression of cell wall-modifying enzyme

732

encoding genes at PK and RR stage in tomato fruit. Expression values are relative to MG

733

stage control. Error bars represent the standard deviation of three biological replicates with

734

three technical replicates each.

735

Figure 2. Heat map of dynamic changes of pectin-modifying enzyme encoding genes to B.

736

cinerea at MG stage (A) and RR stage (B). Columns represent samples collected at different

737

time, and rows represent the different identified genes and relative expression level (Red

738

color means up regulation, green color means down regulation). Expression values are

739

relative to uninfected fruit control at same ripening stage at same interval.

740

Figure 3. Relatvie expression of SlPG1, SlPG2, SlPGIP80.4, SlPME1, SlPME2 and

741

SlPMEI51 in response to B. cinerea in tomato fruit at MG stage (A) and RR stage (B). Gene

742

expression at 6, 12, 24 and 36 h in infected MG fruit and RR fruit was measured using

743

qRT-PCR. Values are relative to the expression of each of the genes in healthy fruit. Star

744

represents fold change (Log based 2) bigger than 1. Error bars represent the standard

745

deviation of three biological replicates with three technical replicates each.

746

Figure 4. Enhancement of polysaccharides of plant cell walls to biological control function

747

of yeast. A, B, Induced resistance of tomato medium cultured R. paludigenum (TMRP)

748

treatment, pectin co-cultured R. paludigenum (PERP) treatment, cellulose co-cultured R. 35



Page 36 of 46

749

paludigenum (CERP) treatment, and xylan co-cultured R. paludigenum (XYRP) treatment

750

for 2 days against B. cinerea in RR stage fruit. After 2 days incubation, new wounds were

751

wounded and inoculated with 30 μL of a B. cinerea suspension. Disease incidence (A) and

752

lesion diameter (B) in tomato fruit were measured 60 h after pathogen inoculation at 25 °C

753

and 90% to 95% RH. C, Population dynamics of TMRP, PERP, CERP and XYRP on tomato

754

fruit surface after inoculation and incubation for 0, 24, 48, 72 and 96 h. D, The dynamic pH

755

values of the fermentation broth of TMRP, PERP, CERP and XYRP in tomato dextrose. E,

756

F, Effect of TMRP, PERP, CERP and XYRP on (E) tube growth (E) and spore germination

757

(F) of B. cinerea evaluated by microscopy after incubation at 25 °C for 18 h. Bars represent

758

standard errors of three replicates. Different letters indicate significant differences (P < 0.05)

759

according to the Duncan's multiple range tests.

760

Figure 5. Polysaccharide-inducible secreted proteins of R. paludigenum and the

761

corresponding fermentation supernatants induce disease resistance in RR stage tomato fruit

762

against B. cinerea. Disease incidence (A) and lesion diameter (B) in polysaccharide-

763

inducible secreted proteins of R. paludigenum, TMRP-P, PERP-P, CERP-P and XYRP-P

764

treatment groups at 48 h after inoculation were inoculated and incubation with pathogen at

765

25 °C for 24 and 36 h. Disease incidence (C) and lesion diameter (D) in TMRP, PERP,

766

CERP and XYRP fermentation supernatants treatment groups at 48 h after inoculation were

767

inoculated and incubation with pathogen at 25 °C for 24 and 36 h. Bars represent standard

768

errors of three replicates. Different letters indicate significant differences (P < 0.05)

769

according to the Duncan's multiple range tests.

770

Figure 6. Enzyme activity of pectin-modifying enzymes and relatvie expression of its 36


Page 37 of 46


771

encoding genes were in response to TMRP-P and PERP-P proteins in tomatoes MG and RR

772

fruit. A, B, Relative expression of pectin-modifying enzymes encoding genes at PK and RR

773

stage in response to TMRP-P (YTSL) and PERP-P (PTSL) in tomato fruit. Expression

774

values are relative to water treated tomato fruit (WTSL) control. Star represents fold change

775

(Log based 2) bigger than 1. C, D, The activities of PG (C) and PME (D) in TMRP-P and

776

PERP-P-treated RR stage fruit wound tissue. Error bars represent the standard deviation of

777

three biological replicates with three technical replicates each. Different letters indicate

778

significant differences (P < 0.05) according to the Duncan's multiple range tests.

37



780

Figure 1

781

A

Page 38 of 46

Maximum force during penetration (N)

40

a

35 30 25 20

b

b

PK Stage

RR Stage

15 10 5 0

MG Stage

Ripening-Stage

B

RR stage

12 8 4 0

*

-4

CEL2

PL8

PPE8B

PMEI61

PMEI51

PMEI22

PME31

PME3

PME2

PME1

PGIL97.3

PGIP26.1

PG2

PG1

-12

PGIP80.4

-8

PG

Log Fold change to MG stage friut (Log base 2)

PK stage

EXP1

782

783

38


Page 39 of 46

784 785


Figure 2 (A) Botrytis cinerea-infected MG stage fruit

(B) Botrytis cinerea-infected RR stage fruit

39


Log Fold change to tissue at 0 hour (Log base 2) 6

4

2

0

-2

12

6

15

10

6

12

6

12

24

-2

-6

12

*

24

-6

24

36

*

24

*

PG 1

* *

5

0

36

PME 1

2

*

0

-2

*

-4

*

*

36

Log Fold change to tissue at 0 hour (Log base 2)

8

PME 1

2

0

*

-4

*

*

36

15

*

10

6

-2

6

6

12

6

12

2

12

*

24

PME 2

2

0

*

* *

-4

* *

12 24

2

24

*

24

36

*

PG 2

6

4

*

0

-2

-4

-6

*

36

PME 2 *

*

0

*

-2

-4

36

ACS Paragon Plus Environment Log Fold change to tissue at 0 hour (Log base 2)

6



PG 1


-8

*


787 10



PG 2

*

*

*

5

0

36


786



Journal of Agricultural and Food Chemistry Page 40 of 46

Figure 3 (A) Botrytis cinerea-infected MG stage fruit PGIP 80.4

4

*

2

0

-2

*

-4

-6

*

6

6

6

2

6

8

6

12

12

*

-8

*

12 24

12

24

24

36

8

*

PMEI 51

*

4

2

0

24 36

(B) Botrytis cinerea-infected RR stage fruit

PGIP 80.4

*

*

*

0

-2

*

36

10

PMEI 51

*

*

6

*

4

*

2

0

36

40

Page 41 of 46


788

Figure 4

789

A

B 36 h after inoculation a

100

36 h after inoculation

48 h after inoculation a

b

Lesion Diameter (mm)

Disease incidence (%)

a

35

b 80

c c

60

d 40

e

d

30

b

25

a

20

c

15

b

P ol P ntr TMR ERP ER YRP P X C Co

d

P ol P ntr TMR ERP ER YRP P C X Co

P ol P ntr TMR ERP ER YRP P X C Co

P ol P ntr TMR ERP ER YRP P C X Co

D a b

a

a

a

b a

0

ab

24

a

b

b a

a

TMRP

PERP

XYRP

CERP

48

72

a

a a

96

Potential of hydrogen in polysccaride mediums (Ph)

C

Yeast population (lg cells per wound)

7.8 7.2 6.6 6.0 5.4 4.8

TMRP

PERP

XYRP

CERP

4.2 0

24

Time after inoculation (Hours)

48

72

96

Fermentation time (Hours)

E

F

a

808 400

80

b

60

b c

40

bc

20 0 -20 -40 k an Bl

RP TM

RP CE

RP PE

RP XY

The pathogen Spore Germination Rate (%)

The pathogen germ tube length (um)

d

0

0

500

d

c 5

790 791 792 793 8.1 794 7957.8 796 7.5 797 7987.2 799 8006.9 8016.6 802 803 804 805 806 807

d

10

d

20


40

100

a

80

b

b

60

40

c

c

RP CE

RP PE

20

0 k an Bl

RP TM

RP XY

41



810

Figure 5

811

A

B

a

a

a

a

ab

90

b

70

c

60 50

a

b

b


c

40 30 20

36 30

b

24

a

18

a

0

a

ab

12

b

6

10

a

a

812 813

PE

R

XY

R

P PC

ER

P PC

o

l ro nt

R TM

P PPE

R

P P-

XY

R

P PC

ER

P P-

C

on

R TM

P P-

on tr o TM l R PPE P R PXY P R PP C ER PP

o

P P-

C

C

l

tr o TM l R PPE P R PXY P R PP C ER PP

0 ro nt

C

Diease incidence (%)

42

b

80

36 h After Inoculation



24 h After Inoculation 100

Page 42 of 46

D





40

100

a

a

a

35

ab

80

ab b

b

b

b

b

60

40

ab abc


Disease incidence (%)

a

30

bc

25

c

20 15 10

20

a b bc

ab b

5

0

0

S Pol -S P-S P-S R ntr TMR ERP ER P XY Co C

-S -S -S -S ol ntr MRP ERP ERP YRP X P T Co C

S Pol -S P-S P-S R ntr TMR ERP ER P XY Co C

-S -S -S ol -S ntr MRP ERP ERP YRP X T P Co C

814

42


c bc

MG Stage Fruit

140

120 a

100

80 b

c

40

20 d

0

RR Stage Fruit

0

* * *

ACS Paragon Plus Environment PG

-8

4 ab

C

5

MG Stage Fruit a

PME3

3

43

D

a

ab

bc

2 c

1

0

RR Stage Fruit

PL8

*

CEL2

MG stage

EXP1

PPE8B

PMEI61

PMEI51

PMEI22

PMEU1

PME31

*

PME2

PME1

PGIP26.1

PGIL97.3

PGIP80.4

PS2

PG2

PG1

-4

**

PE R PP

* *

TM R PP

* * 2

M oc k

* 4

PE R PP

*

TM R PP

12

Log Fold change to YTLS at each stage. (Log base 2)

RR stage

M oc k

*

CEL2

* *

EXP1

*

PL8

*

PPE8B

*

PMEI61

PMEI51

MG stage

PME activity of Solanum lycopersicum under yeast secretomes -1 -1 treatment (umol pectic acid h g FW)

*

PMEI22

** * * *

PE RP -P

60

PMEU1

*

PME3

* **

PME31

**

TM RP -P

-8

* ** *

PME2

*

PME1

*

M oc k

A

PGIL97.3

816

PGIP26.1

Figure 6

PE RP -P

* *

PS2

815

PGIP80.4

4

PG2

-4

PG1

PG

Log Fold change to WTLS at each stage. (Log base 2) 8

TM RP -P

817 818

M oc k

PG activity of Solanum lycopersicum under yeast secretomes -1 -1 treatment (ug D-galacturonic acid h g FW)

Page 43 of 46 Journal of Agricultural and Food Chemistry

B RR stage

6

* *

0

-2

*

-6


819

Page 44 of 46

Table 1 Pectinase activity of Botrytis cinerea with pectinase inhibitor extracts of TMRP-P and PERP-P treated MG and RR stage tomato fruit.

MG stage fruit

RR stage fruit

Mock

TMRP-P

PERP-P

Mock

TMRP-P

PERP-P

Pectinase activity (μg D-galacturonic acid h-1 g-1 Fw)

2899.73±359.99 b

4420.25±571.32 a

3144.62±315.88 b

2654.10±497.14 b

1936.51±352.12 bc

897.63±75.02 c

Inhibitory Percent

35.56%±0.08%

1.77%±0.13%

30.11%±0.07%

41.02%±0.11%

56.97%±0.08%

80.05%±0.02%

820

The inhibitory activity of the pectinase inhibitor was expressed as the percentage reduction in the number of reducing ends liberated by

821

pectinases in the presence of extracted PGIP or PMEI in tomato surface wounds, the activity of B. cinerea pectinase in the presence of inhibitor

822

extraction solution was performed as the control. Different letters indicate signiﬁcant differences (P < 0.05) according to Duncan’s multiple

823

range tests.

44


Page 45 of 46


Graph Abstract

45



Page 46 of 46

SUPPORTING INFORMATION Supporting Information Available: Table 1S. The gene-specific primers of fruit cell wall dissembles related genes in tomatoes, including PMEI22

(XM_004230214.4),

PMEI61

(XM_004234912.4),

PMEI51

(XM_004235032.4),

PPE8B

(XM_004242853.2),

PMEU1

(NM_001246928.2), PME1 (NM_001247222.2), PME2 (NM_001247019.1), PME3 (XM_010326022.3), PME31 (XM_004253289.4), PS2 (NM_001320225.1), PG2 (NM_001247092.2), PG1 (NM_001247906.1), PG (XM_004240049.4), PGIP80.4 (XM_004246280.4), PGIL97.3 (XM_004236497.3), PGIP26.1 (NM_001330726.1), EXP1 (U82123.1), PL8 (XM_004247775.3), CEL2 (NM_001247938.1) and CAC (XM_026032269.1).

46


Depression of Fungal Polygalacturonase Activity in Solanum

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