Secreted Proteins And Effectors Of Marssonina Brunnea F.sp.multigermtubi–the Pathgen Fungus Of Poplar Black Spot Disease

Poplars have emerged as a model system for tree growth and development which hassignificant ecomomic and benefits. During growing and developing periods, poplars will bethreatened by many pathogens. Black spot disease in poplar is a serious leaf disease whichcaused by the fungus, Marssonina brunnea f.sp.multigermtubi. While infecting plant host, fungalpathogens will deploy effectors that contribute to pathogen virulence, and interfere withPTI(PAMP triggered immunity). Study of effectors of M.brunnea, will enhance understandingof the interaction between plant and pathgen. In this study, Marssonina brunneaf.sp.multigermtubi was used as the exprimental material. We separated effectors from secretedproteins and cloned the relational genes, we also predicted and tested effectors from M.brunnea genomedatabase. The main result were as follow:In this study, the secreted proteins of Marssonina brunnea f.sp.multigermtubi were enrichedin artificial liquid medium and separated by two-dimensional polyacrylamide gel electrophoresis.We got50protein spots finally. Peptide sequence of32protein spots were obtained by de-novointerpretion of tandem mass spectra, through the sole use of MS-BLAST, four conservedproteins were identified, including three cell wall degrading enzymes and one fungal elictor.Based on the peptide sequences interpreted from the MS analysis, CODEHOP primers weredesigned to perform cloning genes encoding those secreted proteins. Finally, full-length cDNAsequences encoding15protein spots were obtained. In addition to the four protein spots that werepreviously identified by MS BLAST, we identified11protein spots using this method. Nine ofthese eleven protein had a high degree of sequence similarity, and most of them contain oneLysM domain, which suggested they maybe putative ''LysM effectors''. LysM effectors wereinvolved in sequestering chitin oligosaccharides to prevent elicitation of host immune responses.The other M. brunnea-specific genes did not contain any recognizable domains in their deducedamino acid sequences. They should be other unkown effector.To further explore the traits of the identified genes encoding M. brunnea secreted proteins,we designed specific primers based on the full-length cDNA sequences. Real-time RT-PCR wasemployed to study the expression patterns of these genes during M. brunnea f.sp. multigermtubiS1infection of susceptible poplar clone I-214over a period of6days. To analyze the relationshipbetween gene expression patterns and the infection process, the development of conidia of M.brunnea f.sp. multigermtubi S1on the leaves of clone I-214was monitored by SEM. All threewall-degrading enzyme genes showed same expression patterns that they were induced at4dpi,reaching a maximum level of expression. Most of nine LysM protein genes showed a uniformexpression that they were strongly induced at4dpi and the expression level continued to increaseat6dpi. Observations of scanning electron microscopy showed that germ tubes attempted to penetrate the leaf surface cuticle at1~2dpi, and we observed hyphae within the apoplast spaceof the palisade tissues at4dpi.In order to get more M. brunnea effectors for study, we predicted106putative effectors fromM. brunnea genome database based on the traits of fungi effectors. Using a high-throughputtransient gene expressin system of Populus leaf protoplasts, and using Populus WRKY29as areporter, we developed a workflow to screen fungi effectors which have effect within host plantcell and successfully got one M. brunnea effector that can interfere Populus signaling pathwayinduced by PTI.