Proteomics Provides Insight into the Interaction between Mulberry and

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Proteomics provides insight into the interaction between mulberry and silkworm Dandan Wang, Zhaoming Dong, Yan Zhang, Kaiyu Guo, Pengchao Guo, Ping Zhao, and Qingyou Xia J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 15 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research

Proteomics provides insight into the interaction between mulberry and silkworm

Dandan Wang†, Zhaoming Dong†‡, Yan Zhang†‡§, Kaiyu Guo†, Pengchao Guo†‡, Ping Zhao*†‡, and Qingyou Xia†‡

†State

Key

Laboratory

of

Silkworm

Genome

Biology, ‡Chongqing

Engineering and Technology Research Center for Novel Silk Materials, and §College of Biotechnology, Southwest University, 2 Tiansheng Road, Beibei, Chongqing 400716, China

Corresponding author: Ping Zhao E-mail: [email protected] Tel: +86-23-68251805; Fax: +86-23-68251128;

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 39

ABSTRACT

Mulberry leaves have been selected as food source for the silkworm (Bombyx mori)

over

5000

years.

However,

the

interaction

mechanisms

of

mulberry-silkworm remain largely unknown. In this study, we try to explore the interaction between mulberry and silkworm at the protein level. Total proteins were extracted from mulberry leaves and silkworm feces on day 5 of the fifth larval instar, and analyzed on shotgun liquid chromatography-tandem mass spectrometry, respectively. In total, 2076 and 210 foliar proteins were identified from mulberry leaves and silkworm feces, respectively. These proteins were classified into four categories according to their subcellular location: chloroplast proteins, mitochondrial proteins, secretory-pathway proteins, and proteins of other-locations. Chloroplast proteins accounted for 68.3% in mulberry leaves, but only 23.2% in the feces. In contrast, secretory-pathway proteins had low abundance in mulberry leaves (7.3%) but were greatly enriched to the largest component in the feces (60.1%). Most of foliar secretory-pathway proteins in the feces were found resistant to silkworm feeding by involving in primary metabolite, proteinase inhibiting, cell wall remodeling, redox regulating, and pathogen-resistant processes. On the contrary, only six defensive proteins were identified in the fecal chloroplast proteins including two key proteins responsible for synthesizing jasmanic acid, although chloroplast proteins were the second largest component in the feces. Collectively, the comparative proteomics analyses indicate that mulberry 2


Page 3 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


leaves not only provide amino acids to the silkworm, but display defense against silkworm feeding although the silkworm grows very well by feeding on mulberry leaves, which provides new insights into the interactions between host plant and insect herbivores. Keywords: mulberry leaves; silkworm feces; proteomics; defensive activities;

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 39

1. Introduction

The mulberry leaves have been used as important food crop for the domesticated silkworm for about 5000 years, and the context of mulberry-silkworm interaction has increasingly attracted focus. Efforts have been made to clarify the effect of mulberry foliar compounds on silkworm feeding and growth, such as volatile orders,1 vitamins,2-4 carbohydrates,5,

6

moisture,7, 8 and crude fats.9 However, a growing body of evidence implicate the defensive activities of mulberry leaves against herbivorous insects. For example,

1-deoxynojirimycin

(DNJ),

1,4-dideoxy-1,4-imino-Ɗ-arabinitol

(Ɗ-AB1), adversely affect the growth of lepidopteran insects by inhibiting their gut α-glycosidases;10 tannins tend to form complexes with proteins, starches, and lipids thereby reducing foliar nutrition.11, 12 It has long been known that except for secondary metabolites, plants also mount a defense response by up-regulation of defensive proteins under herbivore attack. Two chitinase-like proteins characterized in the mulberry latex, LA-a and LA-b, show significant insecticidal activities against D. melanogaster.13 MLX56, a mulberry latex protein with two hevein-like chitin-binding domains, shows strong toxicity to lepidopteran caterpillars except for the silkworm.14 Cysteine peptidase inhibitors (cystatins) are abundantly expressed in mulberry leaves,15 and probably play a defense role by inhibiting proteinase activities in herbivorous insect guts.16-18 However, these mulberry defensive compounds seem no toxic to the 4


Page 5 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


silkworm and mechanisms of defense avoidance used in the silkworm have been partly elucidated. The silkworm expressed β-fructofuranosidases instead of α-glycosidases in the gut to circumvent the damage of mulberry sugar-mimic glycosidase inhibitors.19-21 As for mulberry tannins, the high alkalinity of silkworm gut juice may dissociate the complexes between tannins and foliar nutrients;22 additionally, a high ratio of proteins-carbohydrates (2:1~1.7:1) in mulberry leaves would play a positive role in the silkworm to be immune to the deleterious effects of tannins.23-25 Serine proteinases abundantly identified in the silkworm gut, are probably responsible for hydrolysis of mulberry cystatins thereby abolishing their inhibitory effect.15,26 Plants would up-regulate or induce numerous gene expression to defense against insect feeding, which has been supported by a growing body of transcriptome and proteome analysis of susceptible and resistant plants or cultivars against insect feeding.27-30 Mulberry should be no exception when it was attacked by the silkworm. To further explore the interaction of mulberry-silkworm, we extracted and analyzed foliar proteins of mulberry leaves and silkworm feces, respectively. Comparative proteomic analyses showed great reduction in the species and abundance of foliar proteins from mulberry leaves to the silkworm feces, which suggested that a high efficiency of foliar protein digestibility has been evolved in silkworm gut. However, mulberry proteins resistant to silkworm digestion have been identified from silkworm feces at the same time. More than 60% of the fecal proteins were

5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 39

related to biotic stress. Collectively, the comparison of foliar proteins in mulberry leaves and silkworm feces provides novel insights into the interaction between mulberry and silkworm. 2. Materials and methods 2.1. Preparation of mulberry leaves and silkworm feces Silkworms, p50 strain (DaZao), were maintained at the State Key Laboratory of Silkworm Genome Biology at Southwest University of China and reared on mulberry leaves from trees growing at Beibei, Chongqing (29 °N, 106 °E). When larvae reached day 5 of the fifth instar, they were transferred to a new culture vessel and fed using the sixth leaf from the top of the mulberry branch. At the end of day 5, silkworm feces were collected and carefully removed the mulberry leaf debris by a soft brush. Cleaned feces were stored at -80°C until use. Mulberry leaves used for the extraction of proteins were frozen in liquid nitrogen immediately after being picked from the trees, and then stored at -80°C until required for further analysis. 2.2. Preparation of protein samples from mulberry leaves and silkworm feces Mulberry leaves were powdered in liquid nitrogen and suspended in an appropriate volume of extraction buffer (1:3 g/mL) (500 mM Tri-HCl, pH 8.0, including 2% NP-40, 2% MgCl2, 2% β-mercaptoethanol, 1% polyvinyl pyrrolidone, and 1 × inhibitor cocktail (Sigma)). In order to extract as much protein as possible, the suspension was homogenized by sonication for 3 min, then vigorously shaken for 30 min at 4 °C. The suspension was centrifuged

6


Page 7 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(18,000 × g, 20 min, 4 °C), the homogenate was collected, and then proteins were precipitated with cold acetone plus DTT (0.1% w/v) at -30 °C overnight. The precipitate was collected by centrifugation (18, 000 × g, 10 min, 4 °C) and then vacuum-dried (Thermo Savant). The foliar proteins were dissolved in 8 M urea and then used for the analyses. Silkworm feces were soaked in phosphate buffer, pH 7.5 (including 8 g/L NaCl, 0.2 g/L KCl, 2.9 g/L Na2HPO4.12H2O, 0.2 g/L KH2PO4), and then ground in a glass pestle. The fecal suspension was vigorously shaken for 30 min at 4 °C and crude protein extraction was obtained by centrifugation (16,000 × g, 10 min, 4 °C). Non-protein components were removed by adding up to 20% (NH4)2SO4 and proteins were precipitated by further addition of (NH4)2SO4 up to 75%. Fecal proteins was precipitated by centrifugation (16,000 × g, 30 min, 4°C) and dissolved into phosphate buffer; (NH4)2SO4 was removed by dialysis. The proteins were stored at -80 °C and used for further analysis. 2.3. Protein digestion and LC-MS/MS The proteins were digested according to the filter aided sample preparation method.31,32 Briefly, protein solutions (250 µg) were placed in an ultrafiltration tube (MWCO 10,000, Millipore), reduced with 10 mM dithiotreitol for 1 h at 37 °C, then alkylated with 50 mM iodoacetamide for another 1 h in the dark. After being washed three times with 8 M urea and then five times with 50 mM NH4HCO3 in the ultrafiltration tube, proteins were incubated with trypsin (5 µg,

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 39

sigma) for 20 h at 37 °C. Tryptic peptides were collected by centrifugation and concentrated to dryness. Dried peptides were resuspended in 0.1% formic acid and then were separated on the Thermo Fisher Scientific EASY-nLC 1000 system using a Thermo Fisher Scientific EASY-Spray column (C18, 2 µm, 100 Å, 50 µm × 15 cm) with a 180 min acetonitrile gradient (3% ̶ 90%) in 0.1% formic acid at a flow rate of 250 nL/min. The separated peptides were analyzed with a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer. A full MS scan (m/z, ranges of 300-1,800, resolution of 70,000) was acquired using the automatic data-dependent mode of the instrument. The 20 most abundant ions in each full MS were fragmented in the ion trap. A 2 m/z isolation width and 27% relative collision energy were used for fragmentation. The intensity threshold for triggering tandem-MS was set to 1E5. The default charge state of +2 was used. The dynamic exclusion time was 30 s. 2.4. Protein identification and quantification The resulting raw MS data were analyzed with MaxQuant software (version 1.3.0.1).33 The Maxquant searches were executed against a mulberry proteome database containing 25,705 protein sequences, which were downloaded from the Morus genome (http://morus.swu.edu.cn/morusdb/). Peptide searches were performed with the Andromeda search algorithms.34 Carbamidomethylation of cysteine was set as a fixed modification, and N-terminal protein acetylation and methionine oxidation were set as variable

8


Page 9 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


modifications. The minimal peptide length was set to six amino acids, and up to two miscleavages were allowed. The false discovery rate was set to 0.01 for both peptides and proteins. The protein table was filtered to eliminate the identifications from the reverse database and common contaminants. A minimum of one unique peptide was required for an identified protein. The identified peptides and proteins are listed in Supplementary Tables S2-S4. For comparison of the protein abundances, we used the intensity-based absolute quantification (iBAQ) algorithm in the MaxQuant. The estimates of protein intensity are presented in Supplementary Table S2 and S3. 2.5. Protein localization The subcellular localization of foliar proteins was predicted by TargetP according

to

their N-terminal

presequences

(http://www.cbs.dtu.dk/services/TargetP/).35,36 and assigned to four categories: CP

(chloroplast

protein),

MP

(mitochondrial

protein),

SP

(the

secretory-pathway protein) and proteins of other location. 2.6. Incubation experiments of fecal proteins with silkworm digestive juice proteins Fecal proteins (3.8 µg/µL) were dissolved in phosphate buffer, pH 7.5 (prepared as described above) and incubated with digestive juice proteins (3.9 µg/µL) in Britton-Robinson buffer,37 pH 11.0, at 25 °C for different times. The reactions were stopped by the addition of 5 × SDS-PAGE loading buffer and by boiling for 5 min, and then analyzed by SDS-PAGE.

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 39

To obtain the active and non-modified mulberry leaf proteins, mulberry leaves were powdered in liquid nitrogen and suspended in an appropriate volume of extraction buffer (1:3 g/mL) (100 mM Tri-HCl, pH 8.0, including 2% NP-40, 2% sodium L-ascorbate, 2% polyvinyl pyrrolidone, and 5% glycerol), and proteins were extracted using the same method as fecal proteins by being voilently shaken and (NH4)2SO4 precipitated. Mulberry leaf proteins (4.4 µg/µL) were incubated with gut juice proteins in the same way to fecal proteins. 2.7. Protein identification by MOLDI-TOF/TOF The protein band with the molecular weight of 18 kDa on SDS-PAGE gel was excised, destained in an RNAase–free tube, and dehydrated as described by Wang et al.38 Mass spectrometry was performed using a MALDI-TOF/TOF MS analyzer (AB SCIEX 5800, USA), and the data were evaluated as above described in the section of 2.4. Protein identification and quantification with some modifications. A total of 25,705 protein sequences were employed to construct the Protein Pilot software for peptide mass fingerprint (PMF) analysis using carbamidomethyl (C) as the fixed modification, oxidation (M) as the variable modification, and monoisotopic peptide mass and 0.4 Da of mass tolerance as well as 100 ppm of precursor tolerance. 3. Results 3.1. Identification of proteins of foliar origin in mulberry leaves and silkworm feces.

10


Page 11 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


As silkworms displayed the largest ingesta and feces on day 5 of the fifth instar (Supplementary Fig. S1), we chose this developmental stage for the collection of feces and mulberry leaves, and extracted total proteins, respectively. Proteins were detected using SDS-PAGE (Fig. 1A). Comparison of total proteins in the two samples showed huge differences: two abundant protein bands with molecular weights of 120 and 50 kDa in mulberry leaves disappeared in the feces; in contrast, proteins with molecular weights of 40, 30, 18, and 15 kDa were obviously present at higher levels in the feces. These

proteins

were

analyzed

using

shotgun

liquid

chromatography-tandem mass spectrometry, respectively. In total, 9725 and 818 tryptic peptides of foliar origin were identified from the mulberry leaves and silkworm feces, respectively (Supplementary Table S2). In the combined data sets, 2076 and 210 foliar proteins were identified, respectively (Supplementary Table S3 and S4). In theory, all proteins of foliar origin identified in the feces should be included in the mulberry proteome; however, 55 foliar proteins were found exclusively in the feces (Fig. 1B). One possible explanation is that these foliar proteins had too low abundance in mulberry leaves, which were covered by foliar proteins with high abundance so that they could not be detected by the mass spectrometry in mulberry leaves. 3.2. Classification of proteins of foliar origin in mulberry leaves and silkworm feces. To better understand what function of foliar proteins played in the

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 39

interaction of mulberry-silkworm, they were assigned to four categories according

to

their

subcellular

location

based

on

TargetP

(35)

(http://www.cbs.dtu.dk/services/TargetP/): CPs (chloroplast proteins), MPs (mitochondrial proteins), SPs (the secretory-pathway proteins) and proteins of other-location. In mulberry leaves, 646 CPs, 290 SPs, 233 MPs, and 907 proteins of other-location were identified (Fig. 2A). We found that CPs accounted for 68.3% of the total amount of proteins, while SPs, MPs, and proteins of other-location were 7.3%, 5.3%, and 19.1%, respectively (Fig. 2C). Obviously, CPs were the most abundant category in mulberry leaves, which is consistent with the fact that chloroplasts are the center where plant primary metabolic activities take place. Proteins of foliar origin in silkworm feces were analyzed in the same way. Of 210 foliar proteins, 54 CPs, 61 SPs, 17 MPs, and 78 proteins of other-location were identified (Fig. 2B). We found that SPs accounted for more than 60% of the total protein content, while CPs, MPs, and proteins of other-location were 23.2%, 3.3%, and 13.4%, respectively (Fig. 2D). Considering the great reduction of species and abundance of foliar proteins from mulberry leaves to silkworm feces, we concluded that a high efficiency of protein digestibility has evolved in silkworm gut during the long-term interaction of mulberry-silkworm. 3.3. Secretory-pathway proteins of foliar origin in the feces play important antifeedant roles in mulberry defense.

12


Page 13 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In order to compare the proteins’ stability against silkworm gut juice proteinases between mulberry leaves and silkworm feces, the total proteins of the two samples were incubated with silkworm gut juice proteinases for different times and detected using SDS-PAGE, respectively. We found that mulberry leaf proteins were gradually degraded with increasing incubation time; and that they were nearly complete degradation with increasing gut juice proteins to 30 µg (Fig. 3A). As for fecal total proteins, however, hydrolysis was not detected by SDS-PAGE until gut juice proteins were increased to the mass (30 µg) as two twice much as fecal proteins during the incubation (Fig. 3B). A new protein band with the molecular weight of 18 kDa appeared, which was identified as a degrading polypeptide of a protein of foliar origin with the molecular weight 29.3 kDa (a chloroplast ribosomal protein L32) by MOLDI-TOF/TOF (Table 1). The above results indicated that the proteolytic activities of gut proteinases towards fecal proteins were probably inhibited by unknown components in the feces. Considering that SPs were the most abundant in silkworm feces, we supposed that SPs were with antifeedant bioactivities against silkworm feeding and condensed in the feces. This point was supposed by the functional analysis of fecal SPs. They were categorized into, 1) extracellular matrix proteins (59% of the fecal SP abundance); 2) enzymes (19%); 3) proteinase inhibitors (15%); 4) pathogen-resistant proteins (6%), and 5) other proteins (1%) (Fig. 4A). Extracellular matrix proteins were the most abundant in fecal SPs, including 1

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

auxin-binding

protein

ABP19a-like,

2

Page 14 of 39

epidermis-specific

secreted

glycoproteins, 1 basic blue copper family protein, 3 germin-like proteins, 2 cysteine-rich repeat secretory proteins, 2 proline-rich proteins, 1 extensin family protein, 1 hevein-like preproprotein, 1 bark storage protein A, and 1 auxin-induced in root cultures protein. 2) The second major component in fecal SPs was enzyme, including 7 subtilisin-like serine proteinases and 1 cysteine proteinase, 5 GDSL esterases/lipases, 6 oxidoreductases, 1 ribonuclease III, 1 lipoxygenase

homology

domain-containing

protein

1

(LOXHD1),

1

aminotransferase, and 1 dipeptidyl aminopeptidase-like protein 6. 3) The proteins classified as proteinase inhibitors included 2 trypsin-like proteinase inhibitors, 1 cystatin, and 2 bifunctional α-amylase/subtilisin inhibitors, which accounted for a relatively high abundance (15%). 4) Pathogen-resistant (PR) proteins were made up of 5 glucan endo-1,3-β-glucosidases, 3 chitinases, lysosomal alpha-mannosidase, PR-4, wound-induced protein WIN2 precursor and disease resistance protein RPM1. 5) Other proteins consisted of two proteins of unknown function and two firstly identified foliar proteins in the feces, heme-binding protein 2 and citrate-binding protein-like. These proteins were listed in Table 2 in detail. 3.4. Chloroplast Proteins play a role in the activation of mulberry defense. Although chloroplast proteins were greatly degraded by silkworm gut proteinases, there was a relatively high abundance of chloroplast proteins in the feces. In this category, biotic stress-related proteins only accounted for 3%

14


Page 15 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of the total fecal CP abundance (Fig. 4B), including 2 proteins involved in the synthesis of jasmonic acid, which is an important and conserved anti-biotic phyto-hormone in plant kingdom; and 2 downstream effectors of JA-signaling pathway: basic glucan endo-1, 3-beta-glucosidase and leucine aminopeptidase; 1 protein responsible for H2O2 detoxifying and biosynthesis of aromatic compounds, respectively; and 2 ferritins. Whereas, proteins related to photosynthesis were accounted for 95% of the fecal CP abundance, including 12 proteins of light-harvesting complex, 4 proteins of electron transport chain, 5 proteins responsible for redox balance, 1 isochorismatase enzyme, 1 protein of oxygen-evolving complex, and 1 cysteine desulfurase. Other proteins (2%) were mainly made up of proteins responsible for transcription and translation, besides to 1 cysteine synthase, and 1 protein of unknown function. These results indicate us that chloroplast plays an important role in activation of defense against silkworm feeding but is not the center of defensive activities. These proteins were listed in Table 3 in detail. 4. Discussion Plants acting as passive victims, respond to herbivory with production of toxins and defensive proteins that defense against insect feeding.27,29,30,39-43 However, the plant defensive effectors at protein level remain largely unknown. The present study provides us a comprehensive perspective on plant-insect interaction by comparative proteomic analyses of silkworm feces combined with mulberry leaves. Defensive foliar proteins against silkworm feeding were

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 39

widely identified, and most of them were localized in the extracellular matrix. In fecal chloroplast category, very little defensive proteins were identified except for two key proteins responsible for the synthesis of jasmonic acid. These results indicated that JA-signaling pathway may play an important role in mulberry defense against silkworm feeding. 4.1. Jasmonic acid-signaling pathway is important in mulberry defense against silkworm feeding In silkworm feces, a lot of proteins were discovered as the downstream effectors of JA-signaling pathway, such as proteinase inhibitors,44,45 extracellular

matrix

proteins,

basic

pathogen-resistant

proteins,46

oxidoreductases,39 and enzymes responsible for primary metabolites.47 Considering that upstream proteins responsible for JA synthesis identified in silkworm feces, including LOXHD1,48 plastid-lipid-associated protein, and allene oxide cyclase.3,49,50 we think that JA-signaling pathway plays an important role in mulberry defense against silkworm feeding. In plants, when leaves are attacked by insect feeding, excessive reactive oxygen species51 would be rapidly accumulated in chloroplasts, including singlet oxygen, superoxide anion radicals, and H2O2,52 which would result in lipid peroxidation and then produce jasmonic acid,29,30 which would up-regulate the expression of defensive proteins in leaves. Compared with previous studies on the defense of arabidopsis thaliana,18 tobacco,46 soybean,27 potato,17,53,54 and tomato55 against insect feeding, several

16


Page 17 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


defensive enzymes were firstly discovered in the mulberry leaves, such as tryptophan

aminotransferase,

lysine-specific

demethylase,

dipeptidyl

aminopeptidase, and dihydrolipoyllysine-residue acetyltransferase, and an insoluble cell-wall-bound invertase, β-fructofuranosidase (Table 2 and 3). These proteins may help to broaden our understanding of the defensive mechanisms in plants. 4.2. The interaction between mulberry proteinase inhibitors and silkworm gut proteinases Proteinase inhibitors are relatively abundant in fecal SP category. These proteinase inhibitors may directly interact with digestive enzymes to reduce larval biomass and weight,56,57 and induce hyper-production of insect digestive enzymes, which enhances the loss of sulfur-containing amino acids.50,56 As a result, feeding insects become weak, show stunted growth, and died prematurely.58-60 Plant proteinase inhibitors with multi-domains are usually more resistant to insect proteinases;17 the similar effect could be reached when different types of proteinase inhibitors worked in a synergistic manner, which are more effective in preventing the digestion of foliar proteins.17,61-63 In the present study, three types of mulberry proteinase inhibitors were identified in feces (Table 2), namely trypsin-like proteinase inhibitor, cystatin, and the bifunctional α-amylase/subtilisin inhibitor; these proteins may serve to reduce the digestibility of mulberry foliar proteins in the silkworm gut.

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 39

As specialists that feed on mulberry leaves, silkworms are generally able to circumvent the effects of mulberry proteinase inhibitors. Cystatins and serine proteinase inhibitors are characterized as the most abundant types in the mulberry genome.64 Many cystatins have been characterized at the translational level in mulberry leaves; however, most of these cystatins are degraded by silkworm gut proteinases.15 As serine proteinases are mainly responsible for degrading foliar proteins,26 we suggest that mulberry serine proteinase inhibitors have the potential to inhibit silkworm digestion by binding with gut proteinases.65 How the silkworm circumvents the effects of these proteinase inhibitors remains unclear. Previous studies identified several serine proteinase homologs in silkworm gut, which have high sequence similarity as serine proteinase but mutational active site.26 We suggest that serine proteinase homologs may be important in eliminating the inhibitory effect of serine proteinase inhibitors by preferentially binding with them. We found here that many chymotrypsin-like inhibitors in mulberry leaves, and that much inhibitory effect of these proteins, however, was destroyed by silkworm gut juice (Supplementary Fig S2). Our results indicated that silkworms can circumvent mulberry defenses by expressing gut proteinases that could degrade host plant proteinase inhibitors . 4.3. Photosynthesis-related proteins in feces may result from oxidative modification Although great reduction of abundance and species of CP proteins are

18


Page 19 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


detected from mulberry leaves to silkworm feces (Fig 2), some CPs, especially the photosynthesis-related proteins, are found to be stable against being digested by gut juice proteinase, and contribute to the second largest component in the feces. In this study, we find that these photosynthesis-related proteins in silkworm feces are tend to form either electron capture and transport system, and involved in the redox reaction, in which ROS are abundantly produced.66 So, we suppose that oxidative modification would be an important cause of these fecal CPs anti-digestion against silkworm gut proteinases.67-70 Some chloroplast proteins were also highly characterized in M. sexta feces feeding on tomato leaves,71 but the protein profiles are very different from those in silkworm feces. The abundant defense enzymes threonine deaminase and arginase in crucifers39 were not found in mulberry leaves, which probably indicated that defensive proteins should be vary among different plant genetic backgrounds although the defensive mechanisms are conserved in plant kingdom. Additionally, the physical and chemical parameters for these residual CPs in

the

feces

were

computed

using

ProtParam

tool

(http://web.expasy.org/protparam/), including the instability index (II), aliphatic index, and the content of cysteine (Supplementary Table S5). Of the 55 fecal CPs, 36 proteins were classified as stable since their instability index (II) were computed less than 40.72 Among the 19 unstable proteins, 15 proteins contained at least 2 cysteines, which probably enhanced the stability of

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 39

proteins against proteolysis by forming disulfide bond.73 The higher relative volume of aliphatic side chains (Aliphatic Index) in proteins may be another positive factor for the stability of fecal CPs.74 5. Conclusion The comparative proteomics analyses of mulberry leaves and silkworm feces revealed that JA-signaling pathway play a role in defense against silkworm feeding, which is an importantly conserved defense mechanism in plant kingdom. Chloroplasts, as the major driver of photosynthesis and lipid metabolism, easily accumulate excessive ROS when plant leaves are attacked by insects.52 Overproduction ROS would result in polyunsaturated lipid peroxidation to produce JA, which would up-regulate/induce expression of defensive proteins. In the present study, we found these defensive proteins were mainly localized in the extracellular matrix, including proteins enhancing cell wall architecture, proteinase inhibitors, enzymes responsible for redox balance, degrading proteins, lipids, amino acids, and nucleotides (Fig. 5A). Here, combining with previous studies, we divide foliar proteins into four categories (Fig. 5B), 1) nutritive proteins and 2) digestible defensive proteins, which provide amino acid for silkworm growth and development. 3) modified nutritive proteins and 4) un-digestible defensive proteins, which are not efficiently degraded by silkworm gut proteinases and excreted out of gut. Taken together, the results of the present study showed us that JA-signaling pathway play a role in mulberry defense against silkworm feeding, and that the

20


Page 21 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


extracellular matrix is the place where defense activities take place. The information obtained will be of benefit to the understanding of the defense/defense avoidance mechanisms during the interaction between plant and insect herbivores.

Abbreviations: CP(s), chloroplast protein(s); MP(s), mitochondrial protein(s); SP(s), secretory-pathway protein(s); JA, jasmonic acid; ROS, reactive oxygen species; MP, mulberry leaf proteins; FP, fecal proteins; DJ, silkworm gut digestive juice.

Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 31530071 and 31472154). We appreciate the valuable suggestions offered by the editor and referee.

Supporting Information Supplementary Data. Figure S1. Investigation of the ingesta, body weight and fecal weight of the silkworm (DaZao) day by day during the fifth larval instar. Figure S2. Activity staining of mulberry foliar proteinase inhibitors against chymotrypsin after being incubated with silkworm gut digestive juice. (.docx)

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 39

Supplementary Table S1-S5. Tables showing an explanation of the pie chart and column content in the Supplementary Tables S2, S3, S4; a list of identified peptides of foliar origin from mulberry leaves and silkworm feces; a list of foliar proteins identified in mulberry leaves; a list of proteins of foliar origin identified in silkworm feces. Supplementary Table S5 showing results of the predicted physical and chemical parameters for the residual CPs in the feces by ProParam. (XLSX)

22


Page 23 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

References 1. Mooney, A. C.; Robertson, H. M.; Wanner, K. W., Neonate Silkworm (Bombyx mori) Larvae Are Attracted to Mulberry (Morus alba) Leaves with Conspecific Feeding Damage. Journal of Chemical Ecology 2009, 35, (5), 552-559. 2. Horie, Y.; Ito, T., Vitamin requirements of the silkworm. Nature 1963, 197, (4862), 98-99. 3. Ito, T., Effect of dietary ascorbic acid on the silkworm, Bombyx mori. Nature 1961, 192, 951-952. 4. Kanafi, R. R.; Ebadi, R.; Mirhosseini, S. Z.; Seidavi, A. R.; Zolfaghari, M.; Etebari, K., A review on nutritive effect of mulberry leaves enrichment with vitamins on economic traits and biological parameters of silkworm Bombyx mori L. Invertebrate Survival Journal 2007, 4, (2), 86-91. 5. Ito, T., Nutritive values of carbohydrates for the silkworm, Bombyx mori. Nature 1960, 187, (4736), 527-527. 6. Kato, K., Studies on the Digestion of the Carbohydrates Contained in Mulberry Leaves by Silkworms. Part II. Bulletin of the Agricultural Chemical Society of Japan 1934, 10, (7-9), 102-102. 7. Paul, D.; Rao, G. S.; Deb, D., Impact of dietary moisture on nutritional indices and growth of Bombyx mori and concommitant larval duration. Journal of insect physiology 1992, 38, (3), 229-235. 8. Rahmathulla, V. K.; HIMANTHARAJ, M. T.; SRINIVASA, G.; RAJAN, R. K., Association of moisture content in mulberry leaf with nutritional parameters of bivoltine silkworm (Bombyx mori L.). Actaa Entomologica Sinica 2004, 47, (6), 701-704. 9. Srivastava, S.; Kapoor, R.; Thathola, A.; Srivastava, R. P., Nutritional quality of leaves of some genotypes of mulberry (Morus alba). Int J Food Sci Nutr 2006, 57, (5-6), 305-13. 10. Konno, K.; Ono, H.; Nakamura, M.; Tateishi, K.; Hirayama, C.; Tamura, Y.; Hattori, M.; Koyama, A.; Kohno, K., Mulberry latex rich in antidiabetic sugar-mimic alkaloids forces dieting on caterpillars. Proc Natl Acad Sci U S A 2006, 103, (5), 1337-41. 11. Goldstein, J.; Swain, T., The inhibition of enzymes by tannins. Phytochemistry 1965, 4, (1), 185-192. 12. Pandey, S., Do leaf tannin affect non-mulberry silkworm. Indian Silk 1995, 34, (8), 21-23. 13. Kitajima, S.; Kamei, K.; Taketani, S.; Yamaguchi, M.; Kawai, F.; Komatsu, A.; Inukai, Y., Two chitinase-like proteins abundantly accumulated in latex of mulberry show insecticidal activity. BMC Biochemistr 2010, 11, (6), 1471-2091. 14. Wasano, N.; Konno, K.; Nakamura, M.; Hirayama, C.; Hattori, M.; Tateishi, K., A unique latex protein, MLX56, defends mulberry trees from insects. Phytochemistry 2009, 70, (7), 880-8. 15. Liang, J.; Wang, Y.; Ding, G.; Li, W.; Yang, G.; He, N., Biotic stress-induced expression of mulberry cystatins and identification of cystatin exhibiting stability 23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87

Page 24 of 39

to silkworm gut proteinases. Planta 2015, 242, (5), 1139-51. 16. Lison, P.; Rodrigo, I.; Conejero, V., A novel function for the cathepsin D inhibitor in tomato. Plant Physiol 2006, 142, (3), 1329-39. 17. Orr, G. L.; Strickland, J. A.; Walsh, T. A., Inhibition of Diabrotica larval growth by a multicystatin from potato tubers. Journal of Insect Physiology 1994, 40, (10), 893-900. 18. Delledonne, M.; Allegro, G.; Belenghi, B.; Balestrazzi, A.; Picco, F.; Levine, A.; Zelasco, S.; Calligari, P.; Confalonieri, M., Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance. Molecular Breeding 2001, 7, 35–42. 19. Hirayama, C.; Konno, K.; Wasano, N.; Nakamura, M., Differential effects of sugar-mimic alkaloids in mulberry latex on sugar metabolism and disaccharidases of Eri and domesticated silkworms: enzymatic adaptation of Bombyx mori to mulberry defense. Insect Biochem Mol Biol 2007, 37, (12), 1348-58. 20. Daimon, T.; Taguchi, T.; Meng, Y.; Katsuma, S.; Mita, K.; Shimada, T., Beta-fructofuranosidase genes of the silkworm, Bombyx mori: insights into enzymatic adaptation of B. mori to toxic alkaloids in mulberry latex. J Biol Chem 2008, 283, (22), 15271-9. 21. Nakagawa, K.; Ogawa, K.; Higuchi, O.; Kimura, T.; Miyazawa, T.; Hori, M., Determination of iminosugars in mulberry leaves and silkworms using hydrophilic interaction chromatography-tandem mass spectrometry. Anal Biochem 2010, 404, (2), 217-22. 22. Berenbaum, M., Adaptive significance of midgut pH in larval Lepidoptera. American Naturalist 1980, 138-146. 23. Behmer, S. T., Insect herbivore nutrient regulation. Annu Rev Entomol 2009, 54, 165-87. 24. Wang, W. X.; Yang, H. J.; Bo, Y. K.; Ding, S.; Cao, B. H., Nutrient composition, polyphenolic contents, and in situ protein degradation kinetics of leaves from three mulberry species. Livestock Science 2012, 146, (2-3), 203-206. 25. Sujathamma, P.; Dandin, S., Leaf quality evaluation of mulberry (Morus spp.) genotypes through chemical analysis. Indian Journal of Sericulture 2000, 39, (2), 117-121. 26. Zhao, P.; Wang, G. H.; Dong, Z. M.; Duan, J.; Xu, P. Z.; Cheng, T. C.; Xiang, Z. H.; Xia, Q. Y., Genome-wide identification and expression analysis of serine proteases and homologs in the silkworm Bombyx mori. BMC Genomics 2010, 11, 405. 27. Wang, Y.; Wang, H.; Fan, R.; Yang, Q.; Yu, D., Transcriptome analysis of soybean lines reveals transcript diversity and genes involved in the response to common cutworm (Spodoptera litura Fabricius) feeding. Plant Cell Environ 2014, 37, (9), 2086-101. 28. ArginaseSmith, C. M.; Boyko, E. V., The molecular bases of plant resistance and defense responses to aphid feeding: current status. Entomologia Experimentalis et Applicata 2006, 122, (1), 1-16. 29. Loon, L. C. v.; Rep, M.; Pieterse, C. M. J., Significance of Inducible Defense-related Proteins in Infected Plants. Annu. Rev. Phytopathol. 2006, 44, 24


Page 25 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131

135-162. 30. Walling, L. L., The Myriad Plant Responses to Herbivores. J Plant Growth Regul 2000, 19, (2), 195-216. 31. Wisniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M., Universal sample preparation method for proteome analysis. Nature methods 2009, 6, (5), 359. 32. Dong, Z.; Zhang, W.; Zhang, Y.; Zhang, X.; Zhao, P.; Xia, Q., Identification and Characterization of Novel Chitin-Binding Proteins from the Larval Cuticle of Silkworm,Bombyx mori. Journal of Proteome Research 2016, 15, (5), 1435-1445. 33. Cox, J.; Mann, M., MaxQuant enables high peptide identification rates, individualized ppb-range mass accuracies and proteome-wide protein quantification. Nature biotechnology 2008, 26, (12), 1367-1372. 34. Cox, J.; Neuhauser, N.; Michalski, A.; Scheltema, R. A.; Olsen, J. V.; Mann, M., Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 2011, 10, (4), 1794-805. 35. Emanuelsson, O.; Nielsen, H.; Brunak, S.; Von Heijne, G., Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. Journal of molecular biology 2000, 300, (4), 1005-1016. 36. Kleffmann, T.; Russenberger, D.; von Zychlinski, A.; Christopher, W.; Sjolander, K.; Gruissem, W.; Baginsky, S., The Arabidopsis thaliana chloroplast proteome reveals pathway abundance and novel protein functions. Curr Biol 2004, 14, (5), 354-62. 37. Britton, H. T. S.; Robinson, R. A., Universal buffer solutions and the dissociation constant of veronal. J. Chem. Soc. 1931, 1456-1462. 38. Wang, D.; Zhang, Y.; Dong, Z.; Guo, P.; Ma, S.; Guo, K.; Xia, Q.; Zhao, P., Serine protease P-IIc is responsible for the digestion of yolk proteins at the late stage of silkworm embryogenesis. Insect Biochem Mol Biol 2016, 74, 42-9. 39. Chen, H.; Wilkerson, C. G.; Kuchar, J. A.; Phinney, B. S.; Howe, G. A., Jasmonate-inducible plant enzymes degrade essential amino acids in the herbivore midgut. Proc Natl Acad Sci U S A 2005, 102, (52), 19237-42. 40. Oliver Herde, R. A., Joachim Fisahn, Claus Wasternack, Lothar Willmitzer, and Hugo Pefia-Cortés, Localized Wounding by Heat lnitiates the Accumulation of Proteinase lnhibitor II in Abscisic Acid-Deficient Plants by Triggering Jasmonic Acid Biosynthesis. Plant Physiol. 1996, 112, 853-860. 41. Wang, D.; Pajerowska-Mukhtar, K.; Culler, A. H.; Dong, X., Salicylic acid inhibits pathogen growth in plants through repression of the auxin signaling pathway. Curr Biol 2007, 17, (20), 1784-90. 42. Chen, Z.; Agnew, J. L.; Cohen, J. D.; He, P.; Shan, L.; Sheen, J.; Kunkel, B. N., Pseudomonas syringae type III effector AvrRpt2 alters Arabidopsis thaliana auxin physiology. Proceedings of the National Academy of Sciences 2007, 104, (50), 20131-20136. 43. Felton, G. W., Indigestion is a plant's best defense. Proc Natl Acad Sci U S A 2005, 102, (52), 18771-2. 44. Souza, T. P.; Dias, R. O.; Castelhano, E. C.; Brandao, M. M.; Moura, D. S.; Silva-Filho, M. C., Comparative analysis of expression profiling of the trypsin and 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175

Page 26 of 39

chymotrypsin genes from Lepidoptera species with different levels of sensitivity to soybean peptidase inhibitors. Comp Biochem Physiol B Biochem Mol Biol 2016, 196-197, 67-73. 45. Zavala, J. A.; Patankar, A. G.; Gase, K.; Hui, D.; Baldwin, I. T., Manipulation of endogenous trypsin proteinase inhibitor production in Nicotiana attenuata demonstrates their function as antiherbivore defenses. Plant Physiol 2004, 134, (3), 1181-90. 46. Niki, T.; Mitsuhara, I.; Seo, S.; Ohtsubo, N.; Ohashi, Y., Antagonistic effect of salicylic Acid and jasmonic acid on the expression of pathogenesis –related protein genesin wounded mature tobacco leaves. Plant Cell Physiol. 1998, 39, (5), 500-507. 47. Fowler, J. H.; Narvaez-Vasquez, J.; Aromdee, D. N.; Pautot, V.; Holzer, F. M.; Walling, L. L., Leucine aminopeptidase regulates defense and wound signaling in tomato downstream of jasmonic acid. Plant Cell 2009, 21, (4), 1239-51. 48. Blée, E., Impact of phyto-oxylipins in plant defense. TRENDS in Plant Science 2002, 7, (7), 315. 49. Huang, L.-M.; Lai, C.-P.; Chen, L.-F. O.; Chan, M.-T.; Shaw, J.-F., Arabidopsis SFAR4 is a novel GDSL-type esterase involved in fatty acid degradation and glucose tolerance. Botanical Studies 2015, 56, (1), 33. 50. Shukle, R. H.; Murdock, L. L., Lipoxygenase Trypsin Inhibitor, and Lectin from Soybeans: Effects on Larval Growth of Manduca sexta (Lepidoptera: Sphingidae). Environ. Entumol 1983, 12, (3), 787-791. 51. Sheoran, I. S.; Ross, A. R. S.; Olson, D. J. H.; Sawhney, V. K., Compatibility of plant protein extraction methods with mass spectrometry for proteome analysis. Plant Science 2009, 176, (1), 99-104. 52. Kerchev, P. I.; Fenton, B.; Foyer, C. H.; Hancock, R. D., Plant responses to insect herbivory: interactions between photosynthesis, reactive oxygen species and hormonal signalling pathways. Plant Cell Environ 2012, 35, (2), 441-53. 53. Rivard, D.; Cloutier, C.; Michaud, D., Colorado potato beetles show differential digestive compensatory responses to host plants expressing distinct sets of defense proteins. Arch Insect Biochem Physiol 2004, 55, (3), 114-23. 54. Kombrink, E.; Schröder, M.; Hahlbrock, K., Several “pathogenesis-related” proteins in potato are 1, 3-β-glucanases and chitinases. Proceedings of the National Academy of Sciences 1988, 85, (3), 782-786. 55. Meyer, M.; Huttenlocher, F.; Cedzich, A.; Procopio, S.; Stroeder, J.; Pau-Roblot, C.; Lequart-Pillon, M.; Pelloux, J.; Stintzi, A.; Schaller, A., The subtilisin-like protease SBT3 contributes to insect resistance in tomato. J Exp Bot 2016, 67, (14), 4325-38. 56. Lawrence, P. K., Plant protease inhibitors in control of phytophagous insects. EJB Electronic Journal of Biotechnology 2002, 5, (1), 573-580. 57. Johnston, K. A.; Gatehouse, J. A.; Anstee, J. H., Effects of Soybean Protease Inhibitors on the Growth and Development of Larval Helicoverpa armigera. J. Insect Physiol. 1993, 39, (8), 657-664. 58. Zhao, Y.; Botella, M. A.; Subramanian, L.; Niu, X.; Nielsen, S. S.; Bressan, R. A.; 26


Page 27 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219

Hasegawa, P. M., Two Wound-lnducible Soybean Cysteine Proteinase lnhibitors Have Greater lnsect Digestive Proteinase Inhibitory Activities than a Constitutive Homolog. Plant Physiol 1996, 111, 1299-1 306. 59. Bayes, A.; Comellas-Bigler, M.; Rodriguez de la Vega, M.; Maskos, K.; Bode, W.; Aviles, F. X.; Jongsma, M. A.; Beekwilder, J.; Vendrell, J., Structural basis of the resistance of an insect carboxypeptidase to plant protease inhibitors. Proc Natl Acad Sci U S A 2005, 102, (46), 16602-7. 60. Mosolov, V. V.; Grigor'eva, L. I.; Valueva, T. A., Involvement of Proteolytic Enzymes and Their Inhibitors in Plant Protection Applied Biochemistry and Microbiology 2001, 37, (2), 115-123. 61. Oppert, B.; Morgan, T. D.; Culbertson, C.; Kramer, K. J., Dietary mixtures of cysteine and serine proteinase inhibitors exhibit synergistic toxicity toward the red flour beetle, Tribolium castaneum. Comparative Biochemistry and Physiology Part C: Comparative Pharmacology 1993, 105, (3), 379-385. 62. Michaud, D.; Bernier-Vadnais, N.; Overney, S.; Yelle, S., Constitutive expression of digestive cysteine proteinase forms during development of the colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Insect Biochemistry and Molecular Biology 1995, 25, (9), 1041-1048. 63. Broadway, R. M., Are insects resistant to plant proteinase inhibitors? Journal of Insect Physiology 1995, 41, (2), 107-116. 64. He, N.; Zhang, C.; Qi, X.; Zhao, S.; Tao, Y.; Yang, G.; Lee, T.-H.; Wang, X.; Cai, Q.; Li, D., Draft genome sequence of the mulberry tree Morus notabilis. Nature communications 2013, 4, 2445. 65. Potempa, J.; Korzus, E.; Travis, J., The Serpin Superfamily of Proteinase-Inhibitors - Structure, Function, and Regulation. Journal of Biological Chemistry 1994, 269, (23 ), 15957-15960. 66. Unal, D., Effect of Abiotic Stress on Photosystem I-Related Gene Transcription in Photosynthetic Organisms. 2013, 186, (8), 804-804. 67. Bi, J. L.; Felton, G. W.; Mueller, A. J., Induced resistance in soybean toHelicoverpa zea: Role of plant protein quality. Journal of Chemical Ecology 1993, 20, (1), 183-198. 68. Pohnert, G.; Jung, V.; Haukioja, E.; Lempa, K.; Boland, W., New fatty acid amides from regurgitant of Lepidopteran (Noctuidae, Geometridae) caterpillar. Tetrahedron 1999, 55, (37), 11275-11280 69. Møller, I. M.; Kristensena, B. K., Protein oxidation in plant mitochondria as a stress indicator. Photochem. Photobiol. Sci 2004, 8, 730-735. 70. EL-Khavaga, O. Y.; Abou-Seif, M. A. M., Biochemical studies on antioxidant and oxidant activities of some plant extracts. European Review for Medical and Pharmacological Sciences 2010, 14, 731-738. 71. Chen, H.; Gonzales-Vigil, E.; Wilkerson, C. G.; Howe, G. A., Stability of Plant Defense Proteins in the Gut of Insect Herbivores. Plant Physiology 2007, 143, (4), 1954-1967. 72. Gasteiger, E.; Hoogland, C.; Gattiker, A.; Duvaud, S.; Wilkins, M. R.; Appel, R. 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

220 221 222 223 224 225 226 227 228

Page 28 of 39

D.; Bairoch, A., Protein Identification and Analysis Tools on the ExPASy Server. (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press 2005, 571-607. 73. Betz, S. F., Disulfide bonds and the stability of globular proteins. Protein Science A Publication of the Protein Society 1993, 2, (10), 1551. 74. IKAI, A., Thermostability and Aliphatic Index of Globular Proteins. J. Biochem 1980, 88, 1895-1898.

229 230

Figure Legends

231

Figure 1. (A) Proteins extracted from mulberry leaves (lane 1) and silkworm

232

feces (lane 2) were analyzed by SDS-PAGE using 12% gel; (B) Venn diagram

233

illustrates the overlap of foliar proteins from the leaves and feces.

234

Figure 2. Prediction of protein localization based on TargetP. The bar chart A

235

and B represent protein number from each category in mulberry leaves and

236

silkworm feces, respectively; pie charts C and D show the relative abundance

237

of proteins from each category in mulberry leaves and silkworm feces,

238

respectively.

239

Figure 3. Protein degradation profiles detected on SDS-PAGE using 15% gels.

240

(A) Digestion profiles of total fecal proteins (15 µg) incubated with silkworm

241

gut juice proteins (12 µg, and 30 µg, respectively) for different times.

242

Hydrolysis of fecal proteins was not detected on the SDS-PAGE until the gut

243

juice proteins increased to 30 µg. (B) Digestion profiles of total mulberry leaf

244

proteins (30 µg) incubated with silkworm gut juice proteins under the same

245

incubation condition with fecal proteins. The hydrolysis of mulberry leaf

28


Page 29 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


246

proteins became nearly complete with the increase of gut juice proteins. DJ,

247

silkworm gut digestive juice proteins; FP, fecal proteins; MP, mulberry leaf

248

proteins; Arabic numerals, incubation time; arrowhead, a representative fecal

249

protein band.

250

Figure 4. Functional categories of proteins of foliar origin localized to the

251

secretory-pathway (A) and the chloroplast (B) in the silkworm feces.

252

Figure 5. Model describing the major defense activities in mulberry leaves

253

against silkworm feeding. (A) When leaves were attacked by the silkworm,

254

ROS levels increase in chloroplasts, and cause polyunsaturated fatty acid

255

peroxidation to release JA. On the one hand, the products of ROS and lipid

256

peroxidation would react with proteins, thereby reducing the nutritive quality

257

of the leaves. On the other hand, JA-signaling pathway would up-regulate and

258

induce the expression of defensive proteins in the extracellular matrix,

259

including

260

oxidoreductases, and cell wall proteins. (B) In the silkworm gut, nutritive

261

proteins and the degradable defensive proteins were digested and absorbed by

262

silkworm gut. While anti-digested defensive proteins and oxidative proteins

263

were enriched and excreted out silkworm. ROS, reactive oxygen species;

264

Green dot, nutritive proteins; Small green dot, polypeptides or free amino acids;

265

Dark Green dot with black bumps, modified nutritive proteins; Blue dot,

266

digestible proteinase inhibitors; Dark blue dot, un-digestible proteinase

267

inhibitors; Red, digestible defensive enzymes; Dark red, un-digestible

proteinase

inhibitors,

basic

pathogen-resistant

proteins,

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 39

268

defensive enzymes; Yellow aster, digestible oxidoreductases; Dark yellow

269

aster, un-digestible oxidoreductases; Black aster, basic pathogen-resistant

270

proteins; Grey wall, cell wall proteins.

271 272 273 274

Tables. Table 1. Identification of the new protein band (18 kDa) produced during the incubation between fecal proteins and gut juice proteins by GPMAW

275

Protein ID

Corresponding sequence

MW (kDa)

Predicted description

29.3

chloroplast ribosomal protein L32

K·GTSDVEGVVTLTQQDDGPTTVNVR·V Morus011779

R·AFVVHELEDDLGK·G K·GGHELSLTTGNAGGR·L

276 277 278

Table 2. Functional Classification of Fecal Proteins Localized

279

to the Secretory Pathway Category

Proteinase inhibitor

Proteinase

NO.

Description

2

Alpha-amylase subtilisin inhibitors

2

Trypsin-like proteinase inhibitors

1

Cystatin

3

Cucumisin

4

Xylem serine proteinase

1

Cysteine proteinase RD21a

5

GDSL esterase/lipase

Enzyme Lipid degradation

30


Page 31 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Dehydroascorbate reductase

1

Thioredoxin superfamily protein

2

Peroxidases

1

Lipoxygenase homology domain-containing protein 1-like

1

Reticuline oxidase precursor

1

Glutaredoxin

1

Beta-fructofuranosidase, insoluble isoenzyme

5

Glucan endo-1,3-beta-glucosidase

3

Chitinase

1

Lysosomal alpha-mannosidase

1

Tryptophan aminotransferase

1

Dipeptidyl aminopeptidase-like

1

Nuclear fusion defective 2 protein / ribonuclease III

1

Auxin-binding protein ABP19a-like

2

Cysteine-rich repeat secretory protein

3

Germin-like proteins

2

Epidermis-specific secreted glycoprotein

1

Leucine-rich repeat family protein

2

Proline-rich protein

1

Hevein-like preproprotein

1

Bark storage protein A

Oxidoreductase

Glycosylase

Aminotransferase Ribonuclease

Extracellular matrix protein

Pathogen-resistant proteins

Other protein

1

Basic blue copper family protein

5

glucan endo-1,3-beta-glucosidase, basic isoform

3

Chitinase/acidic chitinase

1

Lysosomal alpha-mannosidase

1

Wound-induced protein WIN2 precursor

1

Disease resistance protein RPM1 putative

1

PR-4

1

Heme-binding protein 2

1

Citrate-binding protein-like

2

Uncharacterized

280 281 282

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 39

283 284 285 286 287 288

Table 3. Functional Classification of Fecal Proteins Localized

289

to the chloroplast Category

Description

NO.

Function

oxygen-evolving complex protein 2, 3

1,1

to split water to O2 and 4H+

Light-harvesting complex II protein Lhcb2

1

Chlorophyll a/b binding protein

8

Plastocyanin A

1

Photosystem I P700 chlorophyll a apoprotein A1/ A2

1,1

Photosystem I reaction center subunit II, XI

1,1

2-cysteine peroxiredoxin B

1

to function as light receptors

Glutathione reductase

1

Thioredoxin reductase

1

Ferredoxin

1

Ferredoxin-NADP reductase

1

Fe-superoxide dismutase

1

Ribulose bisphosphate carboxylase small chain

1

Ribulose-phosphate 3-epimerase

1

Fructose-bisphosphate aldolase 1

1

Ribulose bisphosphate carboxylase oxygenase activase 2

1

Carbonic anhydrase

1

Malate dehydrogenase

1

Sedoheptulose-1,7-bisphosphatase

1

Coproporphyrinogen III oxidase

1

oxidative stress Photosynthesis related protein

Calvin cycle

chlorophyll biosynthetic pathways

32


Page 33 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Response to biotic stress

Other proteins

Porphobilinogen deaminase,

1

chlorophyll biosynthetic process

cysteine desulfurase, NifU-like protein 3

1,1

Fe-S cluster formation

Plastid-lipid-associated protein 4

1

jasmonate biosynthesis

Allene oxide cyclase 3

1

jasmonic acid biosynthetic process

shikimate dehydrogenase

1

aromatic compounds biosynthesis

Leucine aminopeptidase 1

1

plant defense

Hydroxyacylglutathione hydrolase 2

1

glutathione biosynthesis

Glucan endo-1,3-beta-glucosidase, basic

1

plant defense

Isochorismatase

1

involving in the biosynthesis of siderophore group

3-hydroxyacyl dehydratase

1

lipid metanolism

Cysteine synthase

1

cysteine formation from serine

DNA repair protein recA homolog 1

1

DNA damage; DNA recombination

Chloroplast stem-loop binding protein of 41 kDa b

1

protein transcription and translation

50S ribosomal protein L6, chloroplastic-like

1


Ribonuclease UK114-like

1


Ferritin

2

storage of Fe

Uncharacterized proteins

4

function of unknown

290

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 39

Figure 1

A kDa 116 66 45 35

M

Mulberry leaves Feces

B

Mulberry leaves

1921

Feces

155

25 18 14


55

Page 35 of 39

Figure 2 Mulberry Leaves

800 600 400 200 0

C

Other CP

SP

MP

Mulberry Leaves 19

.1%

5.3%

B Protein Number

1000

Protein Number

A

7.3%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80

40

CP

20 0

D

Feces

60

MP Other CP

MP

Feces %

.4%

23.2%

SP Other

13

3.3

68.3%

SP

60.1%



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 39

Figure 3 B

A DJ + + + + + + + MP + + + + + + M 0 2 3 4 0 4 (h) 116 66 45 35 25 18 14 kDa

116 66 45 35 25 18 14

DJ + + + + + + + + FP + + + + + + + M 0 2 3 4 0 4 (h)

kDa


B

Page 37 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4 A

1% 6% 15%

19%

Extracellular matrix protein Enzyme Proteinase inhibitor

59%

B

Response to biotic stress Other protein

3% 2% Photosynthesis-related protein Response to biotic stress 95%

Other proteins



Figure 5 A

In chloroplast ROS level increase

nutritive protein oxidation

lipid perioxidation JA synthesis up-regulation accumulation

In extracellular matrix

basic PRs Oxidoreductases cell wall proteins proteinase inhibitors amino acid metabolism

elimination excessive ROS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 39

B absorbed

nutritive proteins degradable defensive proteins

digested enriched

*

*

Midgut

**

d rete nutritive proteins modified by oxidation antidigesited proteins

exc

Hindgut


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


midgut canal

degradable proteins hydrolyzed

* *

insect feeding promoted JA synthesis in choloroplast

up-regulate

d

be

or

bs and a

* enriched* undegradable proteins JA

Page 39 of 39

excreted

in feces

Various defensive and antidigested proteins extracellular matrix


Proteomics Provides Insight into the Interaction between Mulberry and

Recommend Documents