Identification of mycoparasitism-related genes against the phytopathogen Sclerotinia sclerotiorum through transcriptome and expression profile analysis in Trichoderma harzianum.

Andrei Stecca Steindorff,Robert Neil Gerard Miller,Marcelo Henrique Soller Ramada,Alexandre Siqueira Guedes Coelho,Cirano José Ulhoa,Eliane Ferreira Noronha,Georgios Joannis Pappas

doi:10.1186/1471-2164-15-204

Abstract

BackgroundThe species of T. harzianum are well known for their biocontrol activity against plant pathogens. However, few studies have been conducted to further our understanding of its role as a biological control agent against S. sclerotiorum, a pathogen involved in several crop diseases around the world. In this study, we have used RNA-seq and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum gene expression during growth on cell wall of S. sclerotiorum (SSCW) or glucose. RT-qPCR was also used to examine genes potentially involved in biocontrol, during confrontation between T. harzianum and S. sclerotiorum.ResultsData obtained from six RNA-seq libraries were aligned onto the T. harzianum CBS 226.95 reference genome and compared after annotation using the Blast2GO suite. A total of 297 differentially expressed genes were found in mycelia grown for 12, 24 and 36 h under the two different conditions: supplemented with glucose or SSCW. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on SSCW or glucose. We identified various genes of biotechnological value encoding proteins with functions such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. To validate the expression profile, RT-qPCR was performed using 20 randomly chosen genes. RT-qPCR expression profiles were in complete agreement with the RNA-Seq data for 17 of the genes evaluated. The other three showed differences at one or two growth times. During the confrontation assay, some genes were up-regulated during and after contact, as shown in the presence of SSCW which is commonly used as a model to mimic this interaction.ConclusionsThe present study is the first initiative to use RNA-seq for identification of differentially expressed genes in T. harzianum strain TR274, in response to the phytopathogenic fungus S. sclerotiorum. It provides insights into the mechanisms of gene expression involved in mycoparasitism of T. harzianum against S.sclerotiorum. The RNA-seq data presented will facilitate improvement of the annotation of gene models in the draft T. harzianum genome and provide important information regarding the transcriptome during this interaction.

Highlights

The species of T. harzianum are well known for their biocontrol activity against plant pathogens
Illumina sequencing and mapping onto the T. harzianum reference genome In this present study, an RNA-seq approach was used to map genes differentially expressed during T. harzianum growth on S. sclerotiorum cell wall (SSCW)
In our study, a number of genes encoding a serine peptidase, anaspartyl protease, a subtilisin-like, a trypsin-like beyond metallopeptidases (M28, M14) were differentially expressed in the presence of SSCW. These results strongly suggest a role of these enzymes in T. harzianum mycoparasitism against S. sclerotiorum, and support a putative common action mechanism of mycoparasite fungi within the Trichoderma genus

Summary

Introduction

The species of T. harzianum are well known for their biocontrol activity against plant pathogens. Sclerotinia sclerotiorum (Lib.) de Bary is one of the most devastating and cosmopolitan plant pathogens. This fungus infects over 400 species of plants worldwide including important crops and numerous weeds [1]. S. sclerotiorum poses a threat to crops such as sunflower, soybean, oilseed rape, edible dry bean, chickpea, peanut, dry pea, lentils, onion and tulip [1]. It is capable of infecting flowers, leaves, fruits or stems [2] and its life cycle initiates by the germination of sclerotia and formation of infectious propagules. Ascospores are the primary infective propagules of S. sclerotiorum on many crops, and can act in disease scattering since they may be transported to neighboring fields as well as over longer distances [1]

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