Identification And Functional Analysis Of The Differentiation Infection Structure-Specific Genes And Important Kinase Genes In The Wheat Stripe Rust Fungus

Wheat stripe rust,caused by pathogenic fungus Puccinia striiformis f.sp.tritici(Pst)brought serious harm to one of the world’s main crops-wheat.Understanding the pathogenisis of Pst is of great significance to prevention and control of wheat stripe rust.In this study,we functionally characterized differentiation infection structure-specific genes(INF)and kinase genes from Pst at the molecular level.This study will provide gene resources and technical support for sustainable control of wheat stripe rust.Based on the sequence wheat rust genome-wide database in our laboratory,we screened and cloned five genes from Pst associated with infection-structure differentiation.In addition,we selected and cloned 8 putative essential kinase genes from Pst kinome genes.Using real-time RT-PCR,we analyzed the expression patterns of these candidate genes.BSMV-HIGS was used to characterize the function of these candidate genes.The rust hypha length,the number of branches,the number of haustorium,infested area and the shape of spores were observed in the silenced wheat plants.1.Previous studies had proved that there is a kind of specific infection differentiation related genes in Pucciniaceae,only exists in the division of the two taxa-Puccinia and Uromyces.Based on U.appendiculatus three INF genes UaINF8,UaINF24 and UaINF56,using the BLAST analysis,we obtained homologs of INF genes from the genome sequence of Pst and five INF genes from Pst were cloned.Phylogentic analysis indicate that PsINF1,PsINF4 and PsINF11 belong to INF8 family,and PsINF12 and PsINF16 belong to INF24 family.Sequence analysis showed that this five INF proteins without any known function domain or valuable motif.Expression pattern indicated that PsINF1 and PsINF4 transcripts had no obvious difference during the early infection stages.However,PsINF11 and PsINF12 transcripts showed up-regulated continuously at early infection stages and reached the highest at 24 hour post inoculation(hpi).The expression of PsINF16 was up-regulated during Pst infection,whereas the expression peak is at 48 hpi.Furthermore,PsINF11 was silenced by BSMV-HIGS during Pst infection stages.Using barely stripe mosaic virus(BSMV)as a transient expression vector in wheat,the expression of PsINF11 in Pst was suppressed,leading to slower extension of fungal hyphae and reduced production of urediopores.Using scanning electron microscopy(SEM),we analyzed the shape of urediniospores of the Pst on silenced wheat leaves.The results indicated that the urediniospores were dry and flat.These results suggest that the PsINF11 gene might control the stripe rust haustoria formation and play an important role in the differentiation of infection structure of Pst.2.Based on the available genome sequences of Pst in our laboratory,we identified and classified the kinase genes at genome-wide.In total,291 protein kinase(PK)genes were found in the genome of Pst and accounted for about 1%of the whole genome of Pst.All the kinase proteins classified into eleven protein groups,CMGC(57),AGC(50),Alpha(35),CAMK(71),CK1(7),PIKK(14),PDHK(5),TK(1),RIO(6),STE(30)and TKL(14).Based on the essential kinase genes in Fusarium graminearum,twenty putative essential kinase genes in Puccinia striiformis f.sp.tritici were indentified and eight were cloned and designated PstSTE,PstCK2,PstRIO1,PstAKT,PstPKC,PstPIKK,PstAUR and PstCK1,respectviely.Real-time PCR analyses indicated that the expression of PstSTE,PstRIO1,PstPKC,PstPIKK and PstCK1 transcripts were up-regulated significantly during Pst differentiation stages.PstAKT transcripts is relatively higher in alternative host berberis than urediniospore during all infection stages.Knocking down above eight Pst genes by BSMV-HIGS,we found that temporary silencing of PstSTE.PstCK2 and PstCK1 would decrease the number of pustules of Pst significantly,and no urediniopores were produced on wheat leaves carrying PstCK2 and PstCK1 knock-down constructs.These results suggested that these three kinase genes might play important role in regulating Pst development and pathogenicity.