Study On The Function Of GPI-anchored Cell Wall Protein Ss Gsr1 In Regulating The Pathogenicity Of Sclerotinia Sclerotiorum

Rape sclerotinia stem rot,caused by Sclerotinia sclerotiorum,is a significant disease in worldwide rape production.The pathogenesis of this fungus is complex and involves a variety of pathogenic factors including hydrolase,oxalic acid,and secreted proteins.The cell wall is a crucial structure in both plants and pathogenic fungi,consisting largely of chitin and β-glucan.It serves multiple purposes,such as preserving normal cell morphology,withstanding various stresses,as well as detecting and infecting the host.Apart from macromolecular glycans,numerous proteins are anchored to the cell wall by glycosylphosphatidylinositol(GPI)and play an important role.Currently,the role of GPI-anchored cell wall proteins(GPI-CWPs)in S.sclerotinia remains unclear.This study examines the protein SsGsr1 found in S.sclerotinia,which contains both a N-terminal secretory signal and a C-terminal GPI-anchor signal.The intermediate region of the protein is rich in glycine and serine,and is believed to be a GPI-CWP.Through phylogenetic analysis,it was found that homologous proteins of SsGsr1 were mainly found in Ascomycetes,with a concentration in Sclerotinia,Botrytinia and Monilinia.The SsGSR1 gene in S.sclerotinia was knocked out using homologous recombination.While the mycelial morphology and growth rate of the knockout transformants were similar to those of the wild-type strains,the pathogenicity of the knockout transformants was significantly lower for both N.benthamiana and B.napus.This suggests that SsGsr1 plays a role in the pathogenic process of S.sclerotinia.In this study,we utilized protoplast transformation technique to obtain strains expressing HA-SsGsr1 fusion protein.Subsequently,we extracted cytoplasmic protein and cell wall protein from these strains.Western blotting analysis confirmed the localization of SsGsr1 on the cell wall of S.sclerotinia;The hyphae of SsGSR1 gene knockout transformants were examined using transmission electron microscope and Congo red staining.It was discovered that the outer layer of the cell wall was abnormal,and the knockout transformants were more sensitive to cell wall inhibitors.This suggests that SsGsr1 plays a crucial role in maintaining the cell wall integrity of S.sclerotinia mycelium.This study investigated the expression of SsGsr1 in N.benthamiana through Agrobacterium-mediated transient expression.The findings indicate that SsGsr1 induces leaf necrosis in N.benthamiana by targeting the plastid ectodomain,and deletion of the secretory signal prevents this effect.Moreover,the protein was also able to induce leaf necrosis in pepper and tomato,highlighting its broad-spectrum ability to induce cell death.The purified SsGsr1 protein has been shown to trigger the PTI response in plants by causing the accumulation of callose,oxygen burst,and up-regulated expression of defense genes in N.benthamiana.SsGsr1 contains a tandem repeat sequence composed of 10 highly similar repeat units.Further analysis revealed that the 3rd-9th repeat unit was responsible for inducing plant cell necrosis through truncated mutation analysis.Through multiple alignment analysis of homologous sequences,it was discovered that the copy number of tandem repeats varied in SsGSR1 homologous proteins.Specifically,Botrytis cinerea Bc Gsr1 and Monilinia fructicola Mf Gsr1 were both missing 2 and 4 repeat units,respectively.As a result,these proteins lost their ability to induce necrosis.Our study discovered a diverse tandem repeat sequence in the population of Chongqing field rape mycobacterium.Additionally,we observed that certain strains lacked a crucial repeat sequence in the SsGsr1 protein,resulting in the loss of necrosis-inducing activity.This study confirms that SsGsr1,a GPI-CWP of S.sclerotinia,plays a crucial role in maintaining the structure,integrity,and pathogenicity of the cell wall of S.sclerotinia.These findings provide valuable insights into the pathogenic mechanism of S.sclerotinia and pave the way for a deeper understanding of the role of GPI-anchored proteins and tandem repeat sequences in plant pathogenic fungi.