Functional Characterization Of Calcium-binding Proteins PsNCS1 And PsCaMK In Stripe Rust Fungus

Wheat stripe rust,caused by the obligate biotrophic fungus Puccinia striiformis f.sp.tritici(Pst),often imposes a tremendous threat to the production of wheat worldwide.In order to sustainable control of wheat stripe rust,it is paticulary important to understand the molecular mechanisms of pathogenesis of Pst.Currently,cloning and functional analysis of Pst genes,as well as signaling pathway sresearch in Pst have become a hotspot.These studies not only greatly facilitated illustration of the molecular mechanisms of pathogenesis of Pst,but also provided diverse ways for the prevention and control of wheat stripe rust.To further comprehend pathogenesis of Pst and control wheat strip rust,based on screening Pst whole-genome database,we firstly identified Pst geneswhich involved in calcium signaling pathway during infection process of Pst.Their expression patterns and functions were further characterized.1.Based on the Pst whole-genome database in our laboratory,we screened and obtained calcium-binding protein kinase gene PsCaMK which encodsa protein with a protein kinase domain.Real-time RT-PCR analysis indicated that PsCaMK was highly induced in the early stages of Pst infection and peaked at 6 h post inoculation(hpi).When Pst spores were treated with specific immuno suppressor KN-93,their germination rate decreased with the increase of concentration of KN-93.Knocking down the PsCaMK through host-induced gene silencing(HIGS)lead to decrease of infection ability of Pst,significantly inhibition of hyphae length and colony size,and significantly reduction of spore production.2.Based on the bioinformatics analysis of Pst cDNA database,We cloned the ORF regions of gene PsNCSl and its related gene PsPiK1.Real-time RT-PCR showed PsNCS was up-regulated before 48 hpi and has the highest expression in uredispores of Pst.The PsPiK1 transcripts was up-regulated at early infection stages whereas afterwards showed down-regulated.Knocking down the expression of PsNCS1 through BSMV-HIGS,the sporulation ability of Pst reduced,but there is still a considerable number of spores and no significantly inhibition of hyphae length and colony size.However,knocking down the expression of PsPiK1 through BSMV-HIGS lead to reduction of infection ability,significantly inhibition of hyphae length and colony size,and significantly reduction of spore production.Real-time RT-PCR assays indicated knocking down the expression of PsNCS1 lead to the decrease of the expression of PsPiK1 in the early infection stages,especially at12 hpi.These results suggest that PsNCSl may interact with PsPiK1 and took part in Pst pathogenicity.