Functional Study On CsSP3 And CsSP4 Genes Encoding Putitive Secretory Proteins In Bipolaris Sorokiniana

Wheat common root rot is a worldwide soil-borne disease caused by a variety of pathogens,which the components are quite different in different regions.In the Huanghuai Floodplain of China,Bipolaris sorokiniana is one of the main pathogensof this wheat disease.At present,extensive studies have been conducted on the morphology and variation of B.sorokiniana,while understanding of the pathogenesis in molecular level is deficient.Here,we analyzed the role of Cs Sp3 and Cs Sp4 in the pathogenesis by forward genetic method.Our findings would improve understanding on the pathogenic mechanism of B.sorokiniana,which are benefit for the effective prevention and control of B.sorokiniana.Secretory proteins play a very important role in pathogen – host interation.The discoveries ofdetails of secretory proteins from pathogens has greatly promoted our understanding of plant pathogenicity.Through the transcriptome analysis of B.sorokiniana during infection of wheat,four genes with the same expression pattern induced in the early infection stage were identified.Analysis of their protein structures revealed that these all had the typical features of tranditional secreted proteins,including a typical signal peptide structure at the N-terminus and multiple glycosylation sites,serine,threonine and tyrosine phosphorylation sites,which were initially identified as secreted proteins.The four genes were named as Cs SP1(Cochliobolus sativus Secreted Protein gene 1),Cs SP2,Cs SP3,and Cs SP4,respectively.The Cs SP1 deletion mutant was obtained previously with the pathogenicity lost.Therefore,we speculated that the others may also have the similar functions.In this study Cs SP3 and Cs SP4 were chosen for further characterizations.The main results are as follows.Using a reverse genetics approach,a split-marker method was applieded to generate a knockout cassette containing partial fragments of the HYG(hygromycin phosphotransferase)gene with short overlapping region.The hygromycin-resistant transformants are obtained throuth PEG-mediated protoplast transformation and in vivo homologous recombination.(35)cssp3 deletion mutants were primarily screened by PCR amplification and further confirmed by Southern blot analysis.The Cs SP3 encoding region with the native 1.6 kb promoter was amplifed and cloned into the p KNTG vector to make GFP fusion construct at the 3' end.The resulting construct was sequenced and transferred into(35)cssp3 deletion mutant to obtain the complemented transformants.The anaysis results of biological-phynotype showed that(35)cssp3 deletion mutant had no significant differences in colony morphology,colony growth and sporulation compared to wild-type and complemented transformants.Pathogenicity test using conidia to on what andbarley leaves and coleoptile of wheat showed that(35)cssp3 was able to infect,but the lesions formed on the leaves and coleoptiles were significantly reduced,indicating(35)cssp3 involving in pathogenicity of B.sorokiniana.Pot inoculation experiments showed that(35)cssp3 was defect on inhibittion of wheat roots elongation,.The decrease indicates that the Cs SP3 gene is related to the pathogenicity of B.sorokiniana on leaf and stem base of wheat,and may be involved in the secondary metabolism when invading wheat roots,thereby inhibiting the elongation of the host wheat roots.To clarify the subcellular localization of Cs SP3-encoded products,a Cs SP3-GFP overexpression vector driven by a strong RP27 promoter was constructed and transformed into wild-type strain.Through the fluorescence detection of the conidia,germ tube,vegetative mycelium,appressorium and penetration hyphae of onion epidermis simulate infection,we found Cs Sp3-GFP fusion protein was located within the vacuole.Nile red staining further confirmed the colocalization of the Cs Sp3-GFP fusion protein with the lipid droplets in the vacuole.In conidia,germ tubes,and appressoria,most of the fusion proteins were aggregated and fused into large lipid droplets,and the lipid droplets of the invading mycelium were small and dispersed.It is speculated that Cs Sp3 may be related to fat metabolism and glycerol transport to appressor cells to maintain invasive turgor pressure.Taken together,Cs SP3 gene encoding a putative secretory protein with a signal peptide correlates with the pathogenicity of B.sorokiniana on host leaves,and participates in some secondary metabolic processes to inhibit the growth of wheat roots.The fusion protein is located in the vacuole and co-localized with the lipid droplets.It may be related to fat oxidation and product transport,provides energy for the growth of B.sorokiniana and sustains appressoriawith high pressure,which helps to break through the first defense barrier of the host wheat and establish a parasitic relationship.By the same strategy,knockouts of the Cs SP4 gene were successfully obtained,but the mutants didn't differ significantly in terms of colony morphology,growth rate,sporulation and pathogenicity.