Mechanism Of Glycolysis And Early Secretory Pathways Involving In Infection Cushion Formation And Pathogenicity In Sclerotinia Sclerotiorum

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is an important worldwide plant fungal disease.Understanding the mechanisms of sclerotia and infection cushion formation of Sclerotinia sclerotiorum will contribute to the development of new fungicides and plant resistance breeding.In our previous study,the Ss-caf1 gene disruption strain SunfMT6 was unable to form an infection cushion and lost pathogencity to healthy host plants.In this study,we used the wildtype strain Sunf-M and the mutant strain Sunf-MT6 as materials to explore the mechanism of infection cushion formation of S.sclerotiorum through transcriptome,metabolome and gene function studies.The main results are as follows:1.A new metabolic pathway that is associated with infection cushion formation was screened out by comparative transcriptomes and metabolomes analysis:the Glycolytic pathway.By comparing transcriptomes,1216 specially differentially expressed genes(SDEGs)at the early stage(4 h)of infection cushion formation in the strain Sunf-M were screened out,including 661 up-regulated genes and 555 down-regulated genes.We further isolated 91 up-regulated genes with signal peptide from the SDEGs.Gene Ontology(GO)annotation and KEGG pathway enrichment analysis of the up-regulated SDEGs mainly enrich in pathway related to energy metabolism,such as the glycolysis/gluconeogenesis pathway.Particularly,the genes encoding the pyruvate dehydrogenase complex,including SsPDHA and SsPDHB,were significantly up-regulated,while the gene encoding pyruvate decarboxylase,which is involved in another pyruvate degradation pathway,was significantly down-regulated.These results indicate that pyruvate dehydrogenation to acetyl-CoA may play an important role at the early stage of infection cushion formation.Moreover,the metabolome showed that the amount of metabolites targeting to Butanoate pathway,which includes enzymes responsible for acetyl-CoA synthesis,was significantly increased at the infection cushion formation stage of the Sunf-M strain.The content of acetyl-CoA in Sunf-MT6 was 69.6%lower than that in the wildtype.Therefore,the metabolome also implied that the Glycolytic pathway is involved in the infection cushion formation of S.sclerotiorum,and acetyl-CoA is a key metabolite for the infection cushion formation.The above results demonstrate that the transcriptome and metabolome data display a desired correlation,which lays a foundation for deep exploration of the pathogenic genes of S.sclerotiorum.2.Ss-Cafl interacts with the pyruvate dehydrogenase E1 component subunit alpha(SsPDHA)that belongs to the Glycolytic pathway,and SsPDHA is related to infection cushion formation,sclerotia development,and pathogenicity of S.sclerotiorum.The gene SsPDHA is 1413 bp and has 4 exons,encoding a protein with 409 aa and molecular weight of 45.5 kDa.Yeast two-hybrid and pull-down assays showed that Ss-Cafl interacted with SsPDHA.The transcriptome and qPCR analysis showed that SsPDHA was significantly up-regulated at the initial stage of infection cushion formation in Sunf-M but was significantly down-regulated in Sunf-MT6,indicating that SsPDHA is involved in the infection cushion formation and is regulated by Ss-Caf1.Compared with the wildtype,theΔSsPDHA strain exhibited 42.2%decrease of growth rate and was unable to form sclerotia.The density and size of infection cushion formed by the wildtype were 3.6 and 20.4 times than that of the mutants,respectively.These results demonstrate that the phenotypes ofΔSsPDHA were similar to the Ss-caf1 disruption mutant Sunf-MT6.Therefore,it is speculated that Ss-Cafl interacts with SsPDHA to affect the synthesis of acetyl-CoA,and thereby affecting the infection cushion formation and pathgencity of S.sclerotiorum.3.A new pathway that is associated with infection cushion formation was identified:the early secretory pathway,and the genes SsEmp24 and SsErv25 in this pathway were found to be involved in infection cushion formation,sclerotia development,and pathogenicity of S.sclerotiorum.The transcriptome and qPCR analysis showed that the p24 family genes in the early secretory pathway were specifically up-regulated at the early stage of infection cushion formation.Yeast two-hybrid assay revealed that most proteins of the p24 family interacted with each other,and the stable interaction relationship of SsEmp24 and SsErv25 was verified by pull-down assay.Transient expression in Nicotiana benthamiana showed that both SsEmp24 and SsErv25 were located in the endoplasmic reticulum/nuclear envelope.Sequence analysis demonstrated that the gene length of SsEmp24 was 802 bp and the gene had 4 exons,which encoded a transmembrane protein with a length of 203 aa and molecular weight of about 23.1 kDa.The gene SsErv25 was 830 bp and had 4 exons,which encoded a transmembrane protein with a length of 219 aa and molecular weight of about 25.1 kDa.Compared with the wildtype,the ΔSsEmp24 andΔSsErv25 exhibited a slower growth rate with 37.9%and 24.3%decrease,respectively,and both the mutants showed abnormal sclerotia development and declined pathogencity.The density and size of infection cushion formed by wildtype were 5.1 and 4.8 times than that of ΔSsEmp24,respectively,and were 1.29 and 3.16 times than that of ΔSsErv25.respectively.The results demonstrate that SsEmp24 and SsErv25 was involved in the sclerotia development and pathogenicity of S.sclerotia.Moreover,the cellular protein secretion capacity experiment revealed that the protein secretion capacity of ΔSsEmp24 was significantly decreased.Therefore,we speculate that SsEmp24 is the core protein of p24 family and directly interact with SsErv25,then participating in the early secretory pathway to regulate the secretion of proteins,thereby regulating sclerotia development,infection cushion formation,and pathogenicity of S.sclerotiorum.In conclusion,our study identified two new pathogenic related pathways in S.sclerotiorum that are regulated by Ss-Caf1:the early secretory pathway and the Glycolytic pathway.In addition,three pathogenic genes from the two pathways were identified,including SsEmp24 and SsErv25 of the early secretory pathway and SsPDHA of the Glycolytic pathway.All three genes are involved in the regulation of infection cushion formation and sclerotia development,and thus affect the pathogenicity of S.sclerotiorum.Subsequent study can focus on the mechanism of infection cushion formation based on these pathogenic genes,so as to explore the pathogenic mechanism of S.sclerotiorum,and at the same time,it will contribute to a deeper understanding of the mechanism of sclerotinia development.