Functional Characterization Of Genes Involvedin MAPK Signaling Pathway In Stripe Rust And Theirapplication In Wheat Breeding For Rust Resistance

It is evident that obligate biotrophic pathogen Puccinia striiformis f.sp.tritici(Pst)poses a significant threat to wheat production worldwide and jeopardizesglobal food security.Currently,the approaches to manage this disease rely on cultivar resistance coupled with fungicide application.However,driven by a greater need for wheat production,the necessity for environmental protection,the constant evolution of virulence in Pst,and the loss of natural resistance in wheat cultivars,innovative alternative approaches for durable control of wheat stripe rust are required to be established.A powerful genetic tool,RNA interference(RNAi),a conserved eukaryotic mechanism performing a crucial role in gene regulation,has been used to enhance crop resistance by silencing critical genes.Based on this,host-induced gene silencing(HIGS)has been effectively developed and widely used.Mitogen-activated protein kinase(MAPK)cascades regulate a variety of cellular processes in response to extracellular and intracellular stimuli.In this study,we characterized the functions of genes involved in MAPK pathway of Pst.The MAPK kinase gene,PsFUZ7,which was shown to play an important role in Pst virulence by regulating hyphal morphology and development,was selected as RNAi target.These results indicated that the expression of PsFUZ7-RNAi constructs in transgenic wheat plants confers strong and durable resistance to Pst,along with a severe restriction of Pst development.The results were obtained as follows.1.Seven FUS3 / Kss1 cascade candidate genes were obtained by using bioinformatics technique from the genome database of Pst,including PsUBC2(adaptor protein),PsKPP4(MAPKKK homologous gene),PsFUZ7(MAPKK homologous gene),PsKPP2,PsKPP6,PsCRK1(all are MAPK homologous genes),and PsPRF1(transcription factor).qRT-PCR assay showed that six MAPK-relatedgenes were up-regulated at the early stage of Pst infection except PsKPP2,suggesting that the six genes may play an important role in the early stage of infection and mycelial growth effect.Yeast two-hybrid assay indicated that the interaction between PsUBC2-Ps KPP4,PsKPP4-PsFUZ7,PsFU7-KPP6/CRK1 exist,indicating that the interaction of MAPK cascade pathway gene was conserved inPst.Virus-induced gene silencing(VIGS)result indicated that PsKPP4,PsFUZ7,PsKPP6,PsCRK1 and PsPRF1 plays an important role in mycelial growth and development whicheventually leads to a significant inhibition of uredia generation and virulence of Pst.These results strengthen the view that the five genes may have important roles in pathogenesis.In addition,the phenotype of silencing PsFUZ7 was the most significant,suggesting that PsFUZ7 may be at the core of the signal cascade pathway in Pst.2.In this study,we choose Ps KPP4 gene as an important candidate gene to perform further functional analysis.Sequence analysis indicated that Ps KPP4 had typical phosphorylation sites.Yeast two-hybrid assay showed that SAM domain in Ps KPP4 plays an important role in the interaction between PsKPP4 and PsUBC2.Subcellular localization assay,indicated thatthe PsKPP4 was localized in the cytoplasm of wheat protoplasts.PsKPP4 can partially complement the Magnaporthe oryzae mst11 mutant in appressorium formation and plant in fection,indicating that PsKPP4 has some functional conservation among different species.Overexpression of PsKPP4 gene in yeast improved the resistance of yeast cells to external stress compared with the control.In tobacco,expression of PsKPP4 by using the PVX expression system can inhibit the PCD induced by BAX,that is,it may inhibit the HR response of pathogens and plant interactions,indicating that they have the potential to inhibit the plant defense response.VIGS assay showed that silencing of PsKPP4 gene can reduce the pathogenicity of Pst.These results indicated that PsKPP4 is an important pathogenicity factor of Pst,and is involved in the regulation of mycelial growth and pathogenicity.3.We have further found that PsPRF1 protein with HMG domain has a nuclease activity and can induce necrosis in plants and yeasts.Our results suggest that PsPRF1 may regulate its own apoptosis on the one hand and on the other hands may be involved in the degradation of plant DNA.Further experiments are needed to be performed to reveal the underlying mechanism.4.We selected Ps FUZ7 as the key candidate kinase gene for functional analysis.Sequence analysis indicated that PsFUZ7 is a typical MAPK kinase with a conserved phosphorylation site.PsFUZ7 partially complements the Magnaporthe oryzae mst7 mutant in appressorium formation and plant infection,indicating that PsFUZ7 has some functional conservation among different species.Overexpression of PsFUZ7 in fission yeast results in morphological changes and an increase to environmental stress.In accordance with these in vivo data,in vitro assays revealed that stripe rust urediospores treated with the inhibitor germinate at a lower rate and produce a higher frequency of abnormally differentiated and clearly distorted germ tubes or spherical structures along the mycelial apex than controls.VIGS assay revealed that silencing of PsFUZ7 leads to a significant inhibition in mycelialgrowth and development,indicating that PsFUZ7 plays an important role in virulence of Pst.5.To test whether the stable expression of PsFUZ7 silencing constructs can confer resistance to Pst,the PsFUZ7 sequence was introduced into HIGS pAHC25 system,and transformed into Triticum aestivum L.cv.Xinong1376(XN1376)by particle bombardment.The positive plants were identified by molecular biology detection methods such as Southern blot and PCR.Transgenic wheat lines carrying HIGS constructs can produce and process siRNA molecules which efficiently down-regulate PsFUZ7 in Pst,and also affect the expression of some related genes in Pst and wheat.Histological observations showed that the growth of the internode hyphae in the wheat carrying the RNAi vector was strictly limited compared to the control and induced necrosis in the host plant.Bioinformatics analysis was performed to screen similar sequence of PsFUZ7 in wheat stripe rust,wheat,barley and human,in order to avoid off target effect.In addition,12 potential candidates with 1-3 nt mismatches were quantitatively analyzed from the transgenic lines of wheat and Pst.The results showed that the expression of these genes was not significantly down-regulated,and it was proved that the selected sequence is an ideal target for controlling wheat stripe rust.This study indicated that HIGS is a powerful strategy to engineer transgenic wheat resistance against the obligate biotrophic pathogen Pst and has potential as an alternative novel approach todurable control of wheat stripe rust.The application of HIGS in durable control of wheat stripe rust will speed up the wheat germplasm innovation and provides solid theoretical basis and technical support for the control of plant fungal diseases.