Analysis Of Gene Expression Profile In A Negative Immunity Regulator StKH17 Transgenic Potato Plants

RNA-binding proteins(RBPs)have a significant contribution in gene expression regulation,including the expression of defense-associated transcripts involved in plant immunity.RBPs utilize RNA-binding domains(RBDs)that have well-defined structures to create complexes known as ribonucleoproteins(RNPs)that control gene expression by regulating RNA processing,stability,localization,and translation.KH-containing RBPs,which mediate RNA target recognition through the Gxx G motif,have been shown to negatively regulate the plant immunity.St PUB17 is one of the E3 ligases in potato that positively regulates plant defense system by degrading a negative immune regulator,St KH17,in a proteasomedependent manner.St KH17 is an RBP that has previously been implicated as negative potato plant immunity regulator to Phytophthora infestans.Overexpression of St KH17 leads to the enhanced P.infestans colonization and suppression of Cf4/Avr4 mediated cell death in potato.RNA-binding ability of St KH17 has proven to be vital for its function as a suppressor of immunity.To explore the transcriptional changes responsible for the influence of St KH17 on potato immunity,comparative transcriptome analysis was performed between transgenic potato plants overexpressing St KH17(OE-St KH17)and wild-type potato plants.Plants overexpressing St KH17 showed more susceptibility to P.infestans.Based on the result of RNA-seq analysis,222 differentially expressed genes(DEGs)were detected in OE-St KH17 transgenic plants.Functional classification of these DEGs revealed that several genes related to hydrolase activity,monooxygenase activity and integral components of membrane were suppressed.Some genes that have been notably suppressed in expression include GAST1,a protein present in the extracellular region and involved in plant defense,as well as cytochrome P450 and cytochrome b5,which play role in the biosynthesis of flavonoids,thereby contributing to the plant’s defense response.Considering the role of RBPs in post-transcriptional regulation of RNA transcripts,differential analysis of alternative splicing events was conducted.Alternative splicing(AS)analysis revealed that,total of 141 differential alternative splicing events occurred in OESt KH17 transgenic plants.Most genes linked to alternative splicing events comprise phosphatases/kinases,transport related genes,secondary products,and some transcription factors.Our data also indicate some plant resistance-related genes that are being suppressed or alternatively spliced.Significantly suppressed genes include mitogen-activated kinase kinase kinase(Soltu.DM.02G028730),concanavalin A-like lectin protein kinase also known as Lec RK-I.9(Soltu.DM.10G013100)and NB-ARC domain containing protein(Soltu.DM.09G019040)that is responsible for Cf4 mediated hypersensitive cell death response,while three known resistance-related genes being alternatively spliced are serine threonine kinases(STKs).Plants use STKs for downstream signaling to activate mitogen activated protein kinases.All these genes have been reported to be associated with programmed cell death.Interestingly,several defense related genes were upregulated in OE-St KH17 potato plants,including PR1,PR2(glucan endo-1,3-beta-d-glucosidase),PR3(chitinase),osmotin and chalcone isomerase;among these genes PR1 and PR2 are the marker genes of salicylic acid signaling pathway.Upregulation of these defense related genes indicates that St KH17 is somehow negatively regulating immunity.In summary,this study provides novel perceptions about the role of St KH17 in altering the plant defense-response.