Functional Characterization Of Leucine-rich Repeat Receptor-like Kinases(Lrr-rlks) In Development And Pathogenecity Of Phytophthora Capsici

Oomycetes belong to kingdom Chromista and phylogenetically closed to photosynthetic algae.Oomycetes contain around 800 diverse species which consist of many notorious plant pathogens for example,Phytophthora and Pythium genera.P.capsici has been ranked the 5th among the oomycete species which are economically important and threatening the ecosystems P.capsici is a filamentous plant pathogenic oomycete damaging most of the solanaceous crops,legumes and cucurbits worldwide.Living organisms contain receptor protein kinases(RPKs)provide the basic infrastructure to sense,perceive and conduct the signaling events at the cell surface.RPKs have been categorized into various classes on the basis of domains present in the proteins.Leucine-rich repeat receptor-like kinases(LRR-RLKs)being a large class of protein present in several genomes perform the various biological functions related to growth and development,immune responses against biotic/abiotic stress and both.LRR-RLKs are also distributed in oomycetes genome and expressed differentially during infection process.Fisrtly,we carried out a detailed analysis by comparing 14 complete genome sequences from different representative species of four taxonomic groups(Plant,algae,fungi and oomycetes)and LRR-RLKs protein family composed of 111 members has been identified in oomycetes.In oomycetes,LRR-RLKs were found to be independently evolved which were further classified into tweleve subfamilies based on their sequence similarity.The deduced protein sequences of LRR-RLKs identified in all oomycetes species showed the typical structure of an LRR-RLK protein containing LRRs,transmembrane and kinase domains.Some of these families were similar to kinase domains present in plants but few were specific to oomycetes.We identified ten LRR-RLKs present in P.capsici and found that all of the LRR-RLKs were expressed ubiquitously but differently at certain stages of pathogen growth and infection.We selected first three identified LRR-RLKs in P.capsici and characterized their functions in pathogen life cycle.Here,we analyse the role of PcLRR-RK1 in Phytophthora capsici,a model oomycete species and a major plant destroyer of solanaceous and cucurbitaceous vegetable crops.We showed that PcLRR-RK1-silenced P.capsici transformants exhibited reduced growth and produced highly branched fluffy hyphae.The shape and size of sporangia were also altered along with the reduced production of number of sporangia and zoospores.Moreover,silencing of the gene affected the cyst germination and penetration of germ tube into the host tissues,and led to the reduced virulence of P.capsici.Then,we picked the PcLRR-RK2 in Phytophthora capsici and investigated its contribution on growth,reproduction and pathogenecity of P.capsici.Corresponding results obtained showed that PcLRR-RK2 was needed for optimum vegetative growth of P.capsici because the silencing of PcLRR-RK2 resulted in the reduced vegetative growth.Our studies also showed that PcLRR-RK2-silencing cause the defective zoosporogenesis with impaired sporangia production.Our investigation further showed that PcLRR-RK2-silencing also weakens the ability of zoospores germination and penetration into host tissues and failed to produce necrotic lesion.Similarly,the mycelium of silenced transformants also did not penetrate deep into host tissues and showed defects in pathogenecity of P.capsici.Further,we have selected PcLRR-RK3 and characterized its functional importance in P.capsici.PcLRR-RK3-silenced transformants also showed impaired colony growth,decreased deformed sporangia and reduced zoospores count.The mycelium of silenced transformants did not penetrate deep into host tissues and showed defects in pathogenecity of P.capsici.The gene silencing also weaken the ability of zoospores germination and penetration into host tissues and failed to produce necrotic lesion.We also showed that PcLRR-RK3 was localized at the plasma membrane of the cell.Then,we conducted RNA-seq analysis of silenced transformants in all three PcLRR-RLKs(PcLRR-RK1,PcLRR-RK2 and PcLRR-RK3)transformants and found that PcLRR-RLKs-silencing has also affected the differential expression pattern of other genes in P.capsici.Most of DEGs related to growth(annexins,mucins and PLD etc)and virulence(CRN,RxLR,elicitins,CWDE etc)were significantly down-regulated which was validated using qRT-PCR.We also observed that PcLRR-RK1 and PcLRR-RK2 shared more similar DEGs as compared to PcLRR-RK3.Nevertheless,this study outlined the initial picture of the LRR-RLKs distribution,evolution and protein structure in the oomycetes.We further explored by examining the functional characterization of three selected LRR-RLK genes in P.capsici that PeLRR-RLKs(PcLRR-RK1,PcLRR-RK2 and PcLRR-RK3)were required in regulation of vegetative growth,zoospores penetration and establishment into host leaf tissues.