Article pubs.acs.org/jpr
A Bacterial−Fungal Metaproteomic Analysis Enlightens an Intriguing Multicomponent Interaction in the Rhizosphere of Lactuca sativa Marino Moretti,*,†,# Daniela Minerdi,†,¶ Peter Gehrig,‡ Angelo Garibaldi,† Maria Lodovica Gullino,† and Katharina Riedel§ †
Agroinnova - Centre of Competence for the Innovation in the Agro-Environmental Field, University of Torino, Italy Functional Genomics Center, Federal Institute of Technology (ETH) and University of Zurich, Switzerland § Ernst-Moritz-Arndt University of Greifswald & Helmholtz Centre of Infection Research, Institute of Microbiology, Germany ‡
S Supporting Information *
ABSTRACT: Fusarium oxysporum MSA 35 [wild-type (WT) strain] is an antagonistic isolate that protects plants against pathogenic Fusaria. This strain lives in association with ectosymbiotic bacteria. When cured of the prokaryotic symbionts [cured (CU) form], the fungus is pathogenic, causing wilt symptoms similar to those of F. oxysporum f.sp. lactucae. The aim of this study was to understand if and how the host plant Lactuca sativa contributes to the expression of the antagonistic/pathogenic behaviors of MSA 35 strains. A time-course comparative analysis of the proteomic profiles of WT and CU strains was performed. Fungal proteins expressed during the early stages of plant-fungus interaction were involved in stress defense, energy metabolism, and virulence and were equally induced in both strains. In the late phase of the interkingdom interaction, only CU strain continued the production of virulence- and energy-related proteins. The expression analysis of lettuce genes coding for proteins involved in resistance-related processes corroborated proteomic data by showing that, at the beginning of the interaction, both fungi are perceived by the plant as pathogen. On the contrary, after 8 days, only the CU strain is able to induce plant gene expression. For the first time, it was demonstrated that an antagonistic F. oxysporum behaves initially as pathogen, showing an interesting similarity with other beneficial organisms such as mychorrizae. KEYWORDS: metaproteomics, biological control, plant-microbe interaction, antagonistic Fusarium oxysporum, semiquantitative reverse transcriptase PCR
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INTRODUCTION Soilborne pathogenic fungi affecting the health of agricultural plants are the main and constant menace to food production worldwide.1 Among them is the species Fusarium oxysporum which is divided into groups or specialized forms (known as formae speciales) on the basis of the pathogenic ability of strains to cause disease on particular host plants. Some of the formae speciales are further divided into subgroups, named races, on the basis of virulence to a set of different cultivars within the same plant species.2 Management of pathogenic F. oxysporum is difficult because of its wide host range and ability to grow saprophytically or survive for extended periods in the form of thick-walled chlamydospores in the absence of a susceptible crop.3 Fumigation of soil and fungicide applications were the main control strategies for many years, but nowadays, due to the harmful effects of chemicals on the environment and human health, the use of biocontrol has been considered as an appealing and ecological alternative. Use of plant resistant varieties is a practical measure for controlling the © 2012 American Chemical Society
disease in the field, but breeding for resistant varieties using wild genotypes can give undesired outcomes, is time-consuming, and expensive, especially if no dominant gene is known.4 Therefore, microorganisms with antagonistic potential against pathogenic F. oxysporum have been extensively studied and isolated from different environments especially from suppressive soils.5 The nonpathogenic F. oxysporum strains are considered as major biocontrol agents against pathogenic Fusaria.6 Their biocontrol mechanisms include direct antagonism to the pathogen such as competition for nutrients, niches or root colonization, volatileorganic-compounds (VOCs)-mediated antibiosis, and indirect antagonism mediated through the host plant (induced systemic resistance, ISR).6−8 The antagonistic F. oxysporum WT MSA 35 was isolated from naturally suppressive soil in Italy. Efficiency of this strain in reducing severity of Fusarium wilt on different crops has Received: May 10, 2011 Published: February 23, 2012 2061
dx.doi.org/10.1021/pr201204v | J. Proteome Res. 2012, 11, 2061−2077
Journal of Proteome Research
Article
been demonstrated several times.9−11 This strain is actually an association between a pathogenic F. oxysporum belonging to f.sp. lactucae and different rhizobacteria classes attached to the external surface of hyphae.12 The fungus without bacteria, the pathogenic strain called cured (CU) MSA 35, was obtained by antibiotic treatments.12 The virulence silencing of the pathogen and its acquired antagonism depend on the presence of the bacterial consortium.12 The reason this pathogenic fungus lives in intimate association with bacteria is still unknown and under investigation. Proteomics allows for the direct analysis of protein expression and regulation in a specific tissue, cell, or cellular compartment and during various organism interactions. By employing proteomics, difference in the abundance of proteins actually present at the time of sampling can be distinguished and different forms of the same protein can be resolved. Metaproteomics has been defined by Wilmes and Bond as ‘‘the large-scale characterization of the entire protein complement of environmental microbiota at a given point in time.’’13 Metaproteomics is thus the proteome analysis of highly complex biological systems living closely together and functionally interconnected.14 Metaproteomics has emerged as a powerful tool to investigate structural and physiological properties of different phylogenetic groups present in a specific environment or ecosystem such as activated sludge,15 leaf phyllosphere,16 human gastrointestinal tract,17 contaminated soil and groundwater,18 and ocean water.19 In a previous work, we characterized the metaproteome profile of F. oxysporum WT MSA 35 to evaluate the impact of the bacterial consortium on the fungal growth and give insight into the functioning of and the syntrophic interactions within the fungal−bacterial association.20 Specifically, the identification of proteins involved in tissue damage, of both fungal and prokaryotic origin, led us to hypothesize the production of extracellular proteases as possible biocontrol mechanism adopted by F. oxysporum WT MSA 35 to directly face competitors. In addition, many proteins and transporters involved in nutrient uptake may be related to the ability of F. oxysporum WT MSA 35 to compete for nutrients and microelements, a very well described mechanism of biocontrol.21 Moreover, the different regulation of these proteins between CU and WT MSA 35 suggested that the consortium partners extensively exchange nutrients. Probably substances are taken up by bacteria and then translocated to the fungal cells. The aim of the present study was to better define our knowledge on the antagonistic/pathogenic nature of F. oxysporum MSA 35 strains in presence of the host plant Lactuca sativa which can be protected or infected by the two isogenic fungi. In detail, we have evaluated if the plant (together with the bacterial consortium) is able to influence the expression of the antagonistic potential. To this end, F. oxysporum WT and CU MSA 35 were analyzed by comparative two-dimensional gel electrophoresis to detect changes in the protein profiles after 4 and 8 days of growth in presence and absence of lettuce. Microbial infections induce different defense mechanisms in plants, for example, (I) the production of reactive oxygen species (ROS) during the hypersensitive response (HR) to contain pathogen diffusion;22,23 (II) the systemic acquired resistance (SAR) mediated by salicylic acid, in which pathogenesis-related (PR) proteins such as Chitinase and glucanase with antifungal or antibacterial activity are expressed to maintain the resistance state;23,24 and (III) the ISR mediated by jasmonate (JA)/ ethylene pathways which does not involve PR proteins.25
Although our research was mainly focused on fungal reactions elicited by the host plant, the expression analysis of lettuce resistance genes activated during HR, SAR, and ISR in presence of WT and CU MSA 35 was carried out in order to correlate fungal pathogenicity or antagonism with the effective plant response.
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MATERIALS AND METHODS
Fungal Cultures
F. oxysporum MSA 35 WT (carrying the prokaryotic consortium) and CU (the pure fungal culture alone) strains were obtained from the Agroinnova Collection (University of Torino, Italy) grown on Komada’s medium agar26 and kept at 8 °C. For the following experiments, fungi were first grown on potato dextrose broth (PDB; Difco, Detroit, MI) for 15 h at room temperature under slow shaking. Five milliliters of fungal cultures was then plated on potato dextrose agar (PDA; Difco, Detroit, MI) 140 mm plates for 8 days at 27 °C with 12 h of light. Plant Cultivation
Seeds of L. sativa cv White Salad Bowl (coded as 63/2004, Franchi Sementi, Bergamo, Italy) were surface sterilized (2-min 70% ethanol soaking, followed by a 20-min 1% sodium hypochlorite soaking); rinsed (three times) in sterile, distilled water; and placed on Murashige and Skoog medium agar (SigmaAldrich, Steinheim, Germany) in growth cabinets (Sanyo Scientific, Itasca, IL) set to a 12 h of light/12 h of dark cycle under 40-W fluorescent lights. The temperature was maintained at 22 °C with a relative humidity of 80−90%. Germinated seedlings were transferred after 5 days to the plates for the experimental uses described below. Co-culturing of F. oxysporum WT and CU MSA 35−L. sativa
In order to analyze the effect(s) of the host plant on F. oxysporum WT and CU MSA 35 protein profiles, plastic 140 mm dishes containing PDA were used: 5-day-old lettuce seedlings were transferred on the top of half PDA plates, while 2.5 mL of fungal culture grown on PDB was plated in the other half. Plates were then incubated for 4 or 8 days at 27 °C with 12 h of light. Experiments were carried out three times with four replications each and arranged in a completely randomized design. Microscopic Inspections
Lettuce roots were inspected by light microscopy after 4 and 8 days of growth in presence of F. oxysporum WT and CU MSA 35. Roots were washed with 0.6 M MgSO4 and stained with cotton blue (0.1% cotton blue in lactic acid) (Sigma-Aldrich) according to Parija et al.27 Pictures were taken with a Leica DFC425 C photo camera (Leica Microsystems, Mannheim, Germany) mounted on a Zeiss PrimoStar microscope (Carl Zeiss AG, Oberkochen, Germany). Fungal root colonization was estimated after 4 days following Trouvelot et al.28 Briefly, 1 cm-long root pieces stained with cotton blue were mounted on glass slides and observed to evaluate fungal frequency in the root system (F%, defined as number of root fragments surrounded by hyphae/total number of root fragments × 100) and intensity of the fungal colonisation (M%, defined as 95n5 + 70n4 + 30n3 + 5n2 + n1/total number of root fragments, where n5 is the number of fragments rated 5 with a fungal colonization >90%; n4 is the number of fragments 4 with a fungal colonization >50%; n3 is related to a colonization
