Characterization of the induced systemic resistance by Trichoderma spp. in castor

Abstract

Once it was established that Trichoderma strain Th4d colonized castor roots, its ability to trigger induced systemic resistance was studied. Statistically significant reduction of seedling blight caused by Phytophthora as well as wilt caused by Fusarium in Trichoderma treated seedlings against control seedlings indicated to Trichoderema mediated ISR in castor., The next objective was to characterize the ISR using different molecular approaches. The scheme followed and the methods adopted for this purpose are represented in the Fig. 4. Generation of genome-wide transcriptome of castor: Prior to the characterization of ISR at RNA level, it was necessary to establish a good genome-wide transcriptome for castor as only a draft genome sequence of castorbean was available. Utility of draft genome sequence of castorbean is constrained by the limited availability of structural and functional annotation of genes. To ameliorate this deficit, attempt was made towards constructing a near exhaustive genome-wide transcriptome of castorbean. To this end, de novo transcriptome assembly was constructed by RNA-seq from twelve different tissues of castorbean, namely, mature leaf, flower buds (male and female), opened flower (male and female), seedling (two stages), rachis and capsule (four stages) as depicted in Fig. 5. The de novo transcriptome generated has furthered the transcriptome content from the reported 31,221 gene models to 66,601. TRANSCRIPTOME ANALYSIS As per the suggestion in the previous expert committee review meeting, experiments were planned to characterize ISR-boost at transcriptome level: differentially expressed genes and cellular pathways affected post-pathogen-infectionin Th4d treated castor seedlings (compared to mock treated) were identified through RNA-seq. Thus, the pathways altered during ISR boost were identified and this has provided a means to compare ISR-prime and ISR-boost at the gene expression level. This analysis, interestingly, has identified unique as well as common DEGs between ISR-prime and -boost. Validation of representative DEGs through qPCR is in progress. Transcriptome studies of ISR Prime and ISR Boost: Transcriptome studies using the samples of ISR-prime (castor seedlings treated with Trichoderma alone) and ISR-boost (castor seedlings treated with Trichoderma and challenge inoculated with seedling blight pathogen, Phytophthora parasitica var. nicotianae) revealed a statistically significant set of differentially expressing genes across both the conditions i.e. ISR-prime and -boost (Fig.6). Interestingly, only a few sets of genes were identified as commonly regulated between ISR-prime and ISR-boost and similar results have been reported in other systems too. However, further data analysis to identify the pathways differentially regulated in the two conditions has been limited by the large number of unannotated genes that figure in differentially expressed genes in both ISR-prime and ISR-boost. This is being addressed by using different software tools that identify PUFs (proteins of unidentified functions). PROTEOME ANALYSIS To study the effect of Trichoderma-treatment on leaf proteome, 2-Dimensional Gel Electrophoresis (2D) study was undertaken using leaf from untreated castorbean as control. Preliminary studies were indicative of differences between the leaf proteome composition of Trichoderma-treated and -untreated castorbean (Fig.7). Experiments towards verification and characterization of the differences in the proteome content are underway. METABOLOME ANALYSIS

To identify candidate elicitors involved in triggering ISR, analysis of secretome, plant metabolome and Trichoderma secondary metabolites using TLC, HPLC, GC-MS have been adopted. Initial metabolome analyses have shown subtle differences between treated and untreated castor plants. Further analyses of the secretome, metabolome and xylem sap are in progress.

Analysis of Culture Medium (Secretome Characterization) The samples included the aqueous medium from three treatments viz., plant (Ricinus communis) alone, Trichoderma alone and Trichoderma +plant. Plant support medium alone acted as absolute control. Aqueous media (each 50 ml) were extracted successively with n-hexane, ethyl acetate and n-butanol. TLC finger printing was performed on all the extracts. As the n-butanol extract is more polar and expected to accumulate considerable metabolites, it was subjected to LC-MS studies. Analysis of the data revealed that the plant sample contained harzianic acid, whereas only Trichoderma-grown medium showed the presence of phthalide. Trichoderma-treated plant samples contained harzianopyridone, ellagic acid and hydroxyl methyl anthraquinone (Fig.8). HPLC and GC analysis of the secretome samples have also identified different amino acids, organic acids and fatty acid derivatives in the three samples and these will be further confirmed. Plant sample analysis: Root and stem parts of castor plants, treated or untreated with Trichoderma, were air dried after 12 days of growth and these were extracted using different solvents. The extracts were subjected TLC as well as LC. LC analysis of the stem extracts with n-butanol showed presence of a unique compound which is yet to be identified through MS analysis. This is a good lead for further analyses.

Secondary metabolite analysis: Trichoderma strain Th4d grown under stationary condition for one month was subjectedto secondary metabolite analysis and some unique compounds have been discovered but still not confirmed. This will be taken up further so that the secondary metabolites of significance will be identified and explored for further utility.