RNA-mediated silencing of PKS1 gene in Colletotrichum falcatum causing red rot in sugarcane

Red rot is one of the major threatening diseases of sugarcane. Due to frequent breakdown of resistance by simultaneous evolution of pathogen variants with the introduction of newer varieties red rot remains as a challenge for successful breeding programme in India. Earlier studies established that the pathogen has limited genotypic variation with enormous phenotypic variation, which could be mainly due to adaptability of pathogen to the newly released varieties and expression of pathogenicity factors including secondary metabolites viz., toxins, enzymes and pigments like melanin. It is also proved that a positive correlation remains between the production of melanin and disease expression and hence the melanin biosynthesizing genes viz., Polyketide synthase1 (PKS1), Scytalone dehydratase1 (SCD1) and Trihydroxynapthalene reductase1 (THR1) were characterized. In this study, we have utilized RNA interference (RNAi) approach using pSilent-1 vector which facilitates the generation of hair pin constructs that suppress the expression of target gene through Agrobacterium mediated transformation (ATMT) to functionally analyze the major gene PKS1 involved in the production of dihydroxy naphthalene (DHN) melanin to determine its role in virulence in C. falcatum. Functionally active sites of the PKS1 gene i.e., acyl transferase (AT) domain and the region responsible for conidial pigment (CP) polyketide synthase were chosen for knockdown approach. The above said regions were amplified in the virulent C. falcatum isolate (Cf671) and both the sense and antisense fragments were separately cloned into pSilent-1 vector. The expression cassette was then cloned into the binary vector and introduced into Agrobacterium tumefaciens LBA4404 which was later co-cultivated with the fungal spore suspension. Results indicated that the knockdown AT mutant failed to produce spores whereas the CP mutants produced spores as that of the parental strains. Further pathogenicity testing revealed that the disease symptoms of both AT and CP mutants were restricted within the inoculated internode until 15th and 5th dpi respectively while the symptoms of wild type were prominent and crossed beyond 3rd internode at 15 dpi during the host-pathogen interaction. Molecular confirmation of the knockdown mutants with the expression of hygromycin gene and absence of functional domains under RT-PCR clearly indicated that the PKS1 gene has a possible role in C. falcatum pathogenesis. However expression analysis by qRT-PCR indicated regaining of virulence in mutants during later part of host pathogen interaction. The results confirm the applicability of RNAi mediated silencing for functional analysis of pathogenicity gene homologs in C. falcatum.