The Major Components Of SRNA Biogenesis Pathway Act Synergistically To Regulate Pathogenicity And The Activity Of Retrotransposon In Pyricularia Oryzae

Transposition of transposable elements(TEs)results in polymorphisms that facilitate the reshuffling of fungal pathogen genomes.TE mediated polymorphism is also a considerable force that drives the rapid evolution and variations among pathogenesis-related genes,including avirulence(Avr)genes.In eukaryotic cells,transcriptional and post-transcriptional mechanisms,including Small RNAs(s RNAs)associated processes,fine-tune the mobility of retrotransposons.Studies have shown that s RNAs associated post-transcriptional processes crucially promotes genome stability.s RNAs are involved in the inhibition of retrotransposons transposition by facilitating the degradation of transcripts of repetitive elements and also involved in other cellular processes.Previous studies have shown that Po Ago3,Po Rdrp2 and Po Dcl2 play crucial roles in promoting the biogenesis of s RNA in the blast fungus Pyricularia oryzae(syn.Magnaporthe oryzae).Recent studies showed that Po Ago3 and Po Dcl2 inhibit the transposition of an exogenous retrotransposon in the wheat-infecting blast fungus.However,whether the s RNA pathway plays a role in the endogenous regulation of retrotransposons is unknown.In this study,we performed genetic analysis of Po Ago3,Po Rdrp2 and Po Dcl2,the three major components that are responsible for the generation of s RNA in the rice blast fungus to explore:(1)whether Po Ago3,Po Rdrp2 and Po Dcl2 functions synergistically in modulating both asexual development and pathogenesis of the rice blast fungus;(2)whether Po Ago3,Po Rdrp2 and Po Dcl2 acts in association to control the mobility of the retrotransposons in the blast fungus.The knock-out(KO)strains generated in this study were phenotypically analyzed to gain insights on growth rate,germination,appressorium formation,and pathogenicity or virulence characteristics.The result showed a reduction in the vegetative growth ofΔPoago3/ΔPodcl2 and ΔPoago3/ΔPordrp2 double genes deletions strains;deletion of Po AGO3 and Po RDRP2 caused increased sensitivity to the cell wall stressors.The loss of Po DCL2 caused a reduction in conidiation.Furthermore,we investigate the role of these genes in pathogenicity,and the results showed that ΔPoago3/ΔPodcl2 and ΔPoago3/ΔPordrp2 double genes deletion strains showed severely compromised pathogenesis attributes,as they induced smaller lesions on rice leaves and displayed significant defects in the development of invasive hyphae in-planta.Additional comparative analysis of the s RNA sequencing data revealed that the generation of 19-23 nucleotides(nt)s RNAs decreased significantly in the individual mutant strains.Subsequent detailed investigations on the distribution of different sizes of s RNA revealed that the abundance of all the 19-23 nt s RNAs targeted to Copia-1 was lower in all the mutants compared to the wild-type strain.Similarly,the abundance of 19-21 nt s RNAs that target the MGL,RETRO5 and RETRO6 were significantly lower in all the mutants than the abundance recorded for the wild-type strain.These results showed that Po Ago3,Po Rdrp2 and Po Dcl2 contribute to the s RNA biogenesis pathway proteins either through direct or indirect regulation of retrotransposons mechanisms,including Copia-1,MGL,RETRO5 and RETRO6 in the rice blast fungus.Taken together,the results suggested that the s RNA-directed RNAi pathway is involved in the regulation of asexual development,pathogenesis and retrotransposons associated events in P.oryzae.Further studies will be carried out to ascertain whether Po AGO3 could act in tandem with Po DCL2 and Po RDRP2 to regulate the transposition of retrotransposons,such as Copia-1,MGL,RETRO5 and RETRO6.