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Identification of Lens culinaris defense genes responsive to the anthracnose pathogen Colletotrichum truncatum.

ABSTRACT: BACKGROUND: Anthracnose of lentil, caused by the hemibiotrophic fungal pathogen Colletotrichum truncatum is a serious threat to lentil production in western Canada. Colletotrichum truncatum employs a bi-phasic infection strategy characterized by initial symptomless biotrophic and subsequent destructive necrotrophic colonization of its host. The transition from biotrophy to necrotrophy (known as the biotrophy-necrotrophy switch [BNS]) is critical in anthracnose development. Understanding plant responses during the BNS is the key to designing a strategy for incorporating resistance against hemibiotrophic pathogens either via introgression of resistance genes or quantitative trait loci contributing to host defense into elite cultivars, or via incorporation of resistance by biotechnological means. RESULTS: The in planta BNS of C. truncatum was determined by histochemical analysis of infected lentil leaf tissues in time-course experiments. A total of 2852 lentil expressed sequence tags (ESTs) derived from C. truncatum-infected leaf tissues were analyzed to catalogue defense related genes. These ESTs could be assembled into 1682 unigenes. Of these, 101 unigenes encoded membrane and transport associated proteins, 159 encoded proteins implicated in signal transduction and 387 were predicted to be stress and defense related proteins (GenBank accessions: JG293480 to JG293479). The most abundant class of defense related proteins contained pathogenesis related proteins (encoded by 125 ESTs) followed by heat shock proteins, glutathione S-transferase, protein kinases, protein phosphatase, zinc finger proteins, peroxidase, GTP binding proteins, resistance proteins and syringolide-induced proteins. Quantitative RT-PCR was conducted to compare the expression of two resistance genes of the NBS-LRR class in susceptible and partially resistant genotypes. One (contig186) was induced 6 days post-inoculation (dpi) in a susceptible host genotype (Eston) whereas the mRNA level of another ( LT21-1990) peaked 4 dpi in a partially resistant host genotype (Robin), suggesting roles in conditioning the susceptibility and conferring tolerance to the pathogen, respectively. CONCLUSIONS: Data obtained in this study suggest that lentil cells recognize C. truncatum at the BNS and in response, mount an inducible defense as evident by a high number of transcripts (23% of the total pathogen-responsive lentil transcriptome) encoding defense related proteins. Temporal expression polymorphism of defense related genes could be used to distinguish the response of a lentil genotype as susceptible or resistant.

SUBMITTER: Bhadauria V

PROVIDER: S-EPMC3666911 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Identification of Lens culinaris defense genes responsive to the anthracnose pathogen Colletotrichum truncatum.

Bhadauria Vijai V Bett Kirstin E KE Zhou Tengsheng T Vandenberg Albert A Wei Yangdou Y Banniza Sabine S

BMC genetics 20130430

<h4>Background</h4>Anthracnose of lentil, caused by the hemibiotrophic fungal pathogen Colletotrichum truncatum is a serious threat to lentil production in western Canada. Colletotrichum truncatum employs a bi-phasic infection strategy characterized by initial symptomless biotrophic and subsequent destructive necrotrophic colonization of its host. The transition from biotrophy to necrotrophy (known as the biotrophy-necrotrophy switch [BNS]) is critical in anthracnose development. Understanding p ...[more]

PMID: 23631759

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Project description:BackgroundColletotrichum truncatum is a haploid, hemibiotrophic, ascomycete fungal pathogen that causes anthracnose disease on many economically important leguminous crops. This pathogen exploits sequential biotrophic- and necrotrophic- infection strategies to colonize the host. Transition from biotrophy to a destructive necrotrophic phase called the biotrophy-necrotrophy switch is critical in symptom development. C. truncatum likely secretes an arsenal of proteins that are implicated in maintaining a compatible interaction with its host. Some of them might be transition specific.ResultsA directional cDNA library was constructed from mRNA isolated from infected Lens culinaris leaflet tissues displaying the biotrophy-necrotrophy switch of C. truncatum and 5000 expressed sequence tags (ESTs) with an average read of > 600 bp from the 5-prime end were generated. Nearly 39% of the ESTs were predicted to encode proteins of fungal origin and among these, 162 ESTs were predicted to contain N-terminal signal peptides (SPs) in their deduced open reading frames (ORFs). The 162 sequences could be assembled into 122 tentative unigenes comprising 32 contigs and 90 singletons. Sequence analyses of unigenes revealed four potential groups: hydrolases, cell envelope associated proteins (CEAPs), candidate effectors and other proteins. Eleven candidate effector genes were identified based on features common to characterized fungal effectors, i.e. they encode small, soluble (lack of transmembrane domain), cysteine-rich proteins with a putative SP. For a selected subset of CEAPs and candidate effectors, semiquantitative RT-PCR showed that these transcripts were either expressed constitutively in both in vitro and in planta or induced during plant infection. Using potato virus X (PVX) based transient expression assays, we showed that one of the candidate effectors, i. e. contig 8 that encodes a cerato-platanin (CP) domain containing protein, unlike CP proteins from other fungal pathogens was unable to elicit a hypersensitive response (HR).ConclusionsThe current study catalogues proteins putatively secreted at the in planta biotrophy-necrotrophy transition of C. truncatum. Some of these proteins may have a role in establishing compatible interaction with the host plant.

Project description:The ascomycete fungus Colletotrichum truncatum is a major phytopathogen with a broad host range which causes anthracnose disease of chilli. The genome sequencing of this fungus led to the discovery of functional categories of genes that may play important roles in fungal pathogenicity. However, the presence of gaps in C. truncatum draft assembly prevented the accurate prediction of repetitive elements, which are the key players to determine the genome architecture and drive evolution and host adaptation. We re-sequenced its genome using single-molecule real-time (SMRT) sequencing technology to obtain a refined assembly with lesser and smaller gaps and ambiguities. This enabled us to study its genome architecture by characterising the repetitive sequences like transposable elements (TEs) and simple sequence repeats (SSRs), which constituted 4.9 and 0.38% of the assembled genome, respectively. The comparative analysis among different Colletotrichum species revealed the extensive repeat rich regions, dominated by Gypsy superfamily of long terminal repeats (LTRs), and the differential composition of SSRs in their genomes. Our study revealed a recent burst of LTR amplification in C. truncatum, C. higginsianum, and C. scovillei. TEs in C. truncatum were significantly associated with secretome, effectors and genes in secondary metabolism clusters. Some of the TE families in C. truncatum showed cytosine to thymine transitions indicative of repeat-induced point mutation (RIP). C. orbiculare and C. graminicola showed strong signatures of RIP across their genomes and "two-speed" genomes with extensive AT-rich and gene-sparse regions. Comparative genomic analyses of Colletotrichum species provided an insight into the species-specific SSR profiles. The SSRs in the coding and non-coding regions of the genome revealed the composition of trinucleotide repeat motifs in exons with potential to alter the translated protein structure through amino acid repeats. This is the first genome-wide study of TEs and SSRs in C. truncatum and their comparative analysis with six other Colletotrichum species, which would serve as a useful resource for future research to get insights into the potential role of TEs in genome expansion and evolution of Colletotrichum fungi and for development of SSR-based molecular markers for population genomic studies.

Project description:Ascochyta blight of lentil is an important fungal disease in many lentil-producing regions of the world causing major yield and grain quality losses. Quick shifts in aggressiveness of the population of the causal agent Ascochyta lentis mandates developing germplasm with novel and durable resistance. In the absence of complete resistance, lentil genotypes CDC Robin and 964a-46 have frequently been used as sources of partial resistance to ascochyta blight and carry non-allelic ascochyta blight resistance genes. RNA-seq analysis was conducted to identify differences in the transcriptome of CDC Robin, 964a-46 and the susceptible check Eston after inoculation with A. lentis. Candidate defense genes differentially expressed among the genotypes had hypothetical functions in various layers of plant defense, including pathogen recognition, phytohormone signaling pathways and downstream defense responses. CDC Robin and 964a-46 activated cell surface receptors (e.g. receptor like kinases) tentatively associated with pathogen-associated molecular patterns (PAMP) recognition and nucleotide-binding site leucine-rich repeat (NBS-LRR) receptors associated with intracellular effector recognition upon A. lentis infection, and differed in their activation of salicylic acid, abscisic acid and jasmonic acid / ethylene signal transduction pathways. These differences were reflected in the differential expression of downstream defense responses such as pathogenesis-related proteins, and genes associated with the induction of cell death and cell-wall reinforcement. A significant correlation between expression levels of a selection of genes based on quantitative real-time PCR and their expression levels estimated through RNA-seq demonstrated the technical and analytical accuracy of RNA-seq for identification of genes differentially expressed among genotypes. The presence of different resistance mechanisms in 964a-46 and CDC Robin indicates their value for pyramiding gene leading to more durable resistance to ascochyta blight.

Project description:In the present study, Paenibacillus dendritiformis, Trichoderma harzianum, and Trichoderma asperellum were appraised as potential biocontrol agents that induce resistance in chilli (Capsicum annuum) against the devastating pathogen Colletotrichum truncatum, which causes anthracnose. Bright-field and scanning electron micrographs showed the hyphal degradation, lysis, and abnormal swelling in C. truncatum against P. dendritiformis in a dual plate assay. Under greenhouse conditions, chilli seeds pretreated with P. dendritiformis, T. asperellum, T. harzianum, and T. asperellum + T. harzianum by soil soak method inflicted an induced systemic resistance (ISR) in chilli against a C. truncatum-challenged condition. In chilli, the disease index percentage was significantly reduced in the T. asperellum + T. harzianum-treated seeds, followed by the T. harzianum-, T. asperellum-, and P. dendritiformis-treated seeds as compared to the untreated and challenged, respectively. Chilli seeds were primed with T. asperellum + T. harzianum (78.67%), which revealed maximum disease protection under the challenged condition, followed by T. harzianum (70%), T. asperellum (64%), and P. dendritiformis (56%) as compared to untreated and C. truncatum-challenged (6%) condition served as control. The seeds that were pretreated with biocontrol agents (BCAs) inflicted ISR against C. truncatum by enhancing the activity of defence-related enzymes (superoxide dismutase (SOD), peroxidase (POX), polyphenol oxidase (PPO), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and phenylalanine ammonia-lyase (PAL)), accumulating phenolic compounds, and increasing the relative chlorophyll content in chilli. Nitroblue tetrazolium (NBT) and 3,3'-Diaminobenzidine (DAB) stains were used to detect the accumulation of superoxide anion and hydrogen peroxide that appeared nearby the fungal infection sites. The accumulation of reactive oxygen species (O2- and H2O2) in the pathogen-inoculated leaves was a maximum of 48 hpi, followed by P. dendritiformis, T. asperellum, T. harzianum, and T. asperellum + T. harzianum treated tissue upon C. truncatum-challenged condition as compared to the control. Overall, our results showed the potential of T. harzianum, T. asperellum, and P. dendritiformis as biocontrol agents that prevent infection by C. truncatum and inflict an induced systemic resistance in chilli by enhancing the biosynthesis of phenolic compounds, defence and antioxidative enzymes, and reducing the lesion development and reactive oxygen species accumulation. This is the first report of induced systemic resistance against anthracnose in chilli obtained by application of T. harzianum, T. asperellum and P. dendritiformis, through seed priming.

Dataset Information

Identification of Lens culinaris defense genes responsive to the anthracnose pathogen Colletotrichum truncatum.

Publications

Identification of Lens culinaris defense genes responsive to the anthracnose pathogen Colletotrichum truncatum.

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