Screening Of Wheat Stripe Rust Susceptibility Genes And Preliminary Function Analysis Of TaCIPK14-interacting Proteins

Puccinia striiformis f.sp.tritici(Pst)is a kind of obligate parasitic fungus,which seriously affects worldwide wheat production and food security.At present,wheat stripe rust is mainly controlled using fungicides and resistant varieties,but stripe rust has the characteristics of frequent variation and rapid occurrence,so the disease resistance of the existing wheat varieties is weakened or breakdown.Many evidences have shown that the infection and colonization of vegetative parasitic fungi requires the participation of host plant pathogenic factors,and the host plants can obtain a broad spectrum of persistent disease resistance after the deletion and mutation of the susceptible genes.Therefore,identification and functional characterization of susceptible wheat genes is vital for the development of broad-spectrum durable disease resistance.In order to screen and identify stripe rust susceptibility genes in wheat,we analyzed the wheat-Pst interaction transcriptome database,and 24 genes were selected as candidate susceptible genes,which were up-regulated in the compatible combination and down-regulated in the incompatible combination.Among the 24 candidate genes,5 genes including cytochrome P450,hypothetical protein TRIUR3,Bowman-Birk trypsin inhibitors,serine/threonine protein kinase TaEDR1 and 2-oxidation glutaric acid dependent two-plus oxygen enzyme gene negatively regulated the resistance to stripe rust through virus induced gene silencing(VIGS)technology.Spore production quantity and the colony area after silencing these genes were greatly reduced.So,these 5 genes showed a potential susceptibility function to Pst.A wheat gene,TaCIPK14,was identified as a candidate wheat stripe rust susceptibility gene.In order to further analyze the TaCIPK14-mediated regulation mechanism,we identified TaCIPK14-interacting proteins.Seven potential target proteins were obtained by yeast two hybrid library screening technology.Two TaCIPK14 proteins,E3 ubiquitin-protein ligase(TaRCP1)and proteasome subunit beta type-1(TaPBF1)were identified as TaCIPK14-interacting proteins by using yeast two-hybrid and GST pull-down technology.After bioinformatics analysis,we found that the total ORF length of TaRCP1 gene was 897 nucleotides,encoding 298 amino acid residues,including a RING protein domain.The total ORF length of TaPBF1 gene was 666 nucleotides,encoding 221 a mino acid residues,including a PER1 domain.After subcellular localization analysis,we found that TaRCP1 was distributed in the cell membrane,cytoplasm and nucleus,while TaPBF1 protein was aggregated in the cytoplasm,which was speculated to be proteasome according to its annotation function.TaRCP1 and TaPBF1 showed differential expression changes during interaction between wheat and Pst,suggesting that they may play a role during interaction between wheat and Pst.After the silencing of TaRCP1 and TaPBF1 by VIGS,the uredium production were reduced after inoculation with a compatible Pst strain CYR31.Therefore,we speculate that TaCIPK14 may affect the protein degradation process by regulating TaRCP1 and TaPRF1,and thus play a susceptibility role during interaction between wheat and Pst.