Functional Characterization Of Starch Phosphorylase Gene TaPho2 During Wheat-stripe Rust Interaction

Two isoforms of plant starch phosphorylase?Pho1 and Pho2?mainly regulate starch metabolism during the growth and developmental phases of plants.Pho contributes to both the synthesis and degradation of glucan chains by catalyzing a reversible transfer of glucosyl unit from glucose1-phosphate?Glc1P?to the non-reducing end of an?-1,4-linked glucan chain.On the other hand,Puccinia striiformis f.sp.tritici?Pst?,the causal organism of wheat stripe rust is now a serious threat to wheat production and food security worldwide.However,the functions of starch phosphorylase genes in wheat-Pst interaction are still unknown.This study aimed to functionally characterize TaPho2 during the wheat-Pst interaction.Based on Pho sequences from T.aestivum cv.Chinese Spring by Ensemble Plant BLAST searches using Arabidopsis and rice Pho protein sequences as query revealed a total of 9putative Pho sequences were identified in A,B and D sub-genome?3 loci for each sub-genome?of the wheat genome.Phylogenetic analysis showed that Pho protein sequences identified from wheat genome,3 sequences located on chromosomes 5 clusters with Pho1 and6 sequences located on chromosome 3 clusters with Pho2 protein sequences from monocot plants.Multiple sequence alignment revealed that 3 loci located on chromosomes 5A,5B and5D had 98.38%nucleotide sequence identity,while 6 loci located on chromosomes 3A,3B and 3D had 98.57%nucleotide sequence identity.Thus,we concluded that the sequences located on each chromosome were copy of same gene and sequences located on chromosome5 and chromosome 3 were designated as Ta Pho1 and TaPho2 respectively.The relative transcriptions of TaPho2 gene to Pst infections was confirmed by q RT-PCR analysis.TaPho2 was significantly induced in wheat leaves infected by the virulent Pst isolate CYR31,While,no significant relative expression of TaPho2 was observed in the incompatible interaction.Thus,we supposed that TaPho2 may co-related with wheat susceptibility to Pst.To further verify our speculation,TaPho2 gene was suppressed by using a virus-induced gene silencing?VIGS?system to evaluate the function of TaPho2 during the interaction between wheat and Pst.Silencing of TaPho2 increased wheat resistance to Pst by increasing H₂O₂ accumulation to reduce fungal growth in both compatible and incompatible interactions,indicating that TaPho2 is a positive regulator of wheat susceptibility to Pst.To improve our understanding concerning TaPho2 function in programmed cell death?PCD?,TaPho2 was studied against PCD triggered by a mammalian Bax gene and a Pst elicitor-like protein Pst322.Overexpression of TaPho2 suppressed Pst322-or BAX-triggered cell death in wheat and N.benthamiana leaves.In addition,suppression of TaPho2 gene using VIGS system demonstrates that TaPho2 is a negative regulator of plant cell death triggered by Pst through reducing H₂O₂ accumulation.Thus,we speculated that TaPho2 may regulate wheat susceptibility to Pst via ROS-dependent manner.To further confirmation of our results,TaPho2 was demonstrated to specifically interact with wheat catalase3 Ta CAT3 by yeast two-hybrid system,Bi FC,GST pull-down and Co-IP assay.Plant catalase play critical roles in plant development and defense by eliminating excess amounts of H₂O₂ and regulating PCD.Enzyme activity assay of catalase revealed that TaPho2 increased the enzymatic activity of Ta CAT3 in vivo and in vitro.Moreover,the relative expression of Ta CAT3 was significantly reduced in TaPho2-knockdown plants indicating that,TaPho2 may form a complex with Ta CAT3 to regulate H₂O₂mediated PCD in wheat during Pst infections.Taken together,we suggest that TaPho2 regulates H₂O₂homeostasis via interaction with Ta CAT3 and,therefore,promotes Pst infection in wheat.To our knowledge,this is first the evidence indicating that a starch phosphorylase gene may functions as a susceptibility factor in wheat to facilitate Pst infection through interaction with catalase.