Proteomics Of Verticillium Dahliae Secretory Proteins And Structure-based Functional Analyses Of Corresponding Homologs From Saccharomyces Cerevisiae

Verticillium dahliae is a kind of soilborne pathogenic fungi widely distributed in the soil and one of the main causal agents of vascular wilt towards a wide range of plant species worldwide. It can cause the plant Verticillium wilt, leading to the huge economic lost. V. dahliae causes not only external symptoms such as leaf chlorosis and necrosis, but also the internal symptoms such as vascular discoloration of the host plant, and finally makes the plant necrosis and decay. However, we have no way to fight against V. dahlia to date. How V. dahliae invades the plants or its interaction with the host plant remains unclear. The functional research on the key virulence proteins is also very preliminary.It has been reported that nitrogen limiting condition may be one of the stimuli during pathogen invasion, whereas nitrogen limiting of environment mimics this simulation. In addition, under nitrogen limiting conditions, many genes coding for virulence-related elicitor proteins were rapidly induced, and similarly, the nitrogen metabolism genes were induced when the pathogen faced the nutrition block. Nutritional stress, especially nitrogen limiting pressure has also been demonstrated to be the key factor to control the growth and development of pathogenic fungi. In short, when pathogenic fungi invade plants, the internal of the host plant is an environment of nitrogen limiting for fungi. In other words, nitrogen limiting condition can mimic the environment in vivo where the pathogenic fungi invade the plants.Many signaling molecules are involved in the recognition of the pathogen with plants. Fungal secretory proteins play an active role in this process. In many cases, the extracellular secretion of pathogenic factors and elicitor proteins is required for executing their biological functions.The rapidly updated genome sequencing technologies and many accomplished plant pathogenic genomes enable us to identify the genome-wide pathogenic virulence factors. Here we utilized the proteomic approach to investigate the secreted proteome of V. dahliae under nitrogen limiting. Using either the in-gel and direct digestion, combined with the two-dimensional liquid chromatography electrospray ionization tandem-mass spectrometry (2D-LC-MS), we analyzed the secreted proteome of V. dahliae under nitrogen limiting. In total,214proteins have been identified. These proteins include10functional terms related to the cell structure,8functional terms related to the molecular function, and19functional terms related to the biological process, according to sequence analysis and GO(Gene Ontology) gene function annotation. After further analyses,33proteins were identified as secreted proteins under nitrogen limiting. These secreted proteins have an isoelectric point from4.56to9.51, and a molecular weight of13to86kDa. Combined with the sequence analysis and GO function annotation, these secreted proteins can be divided into6categories: elicitor proteins and immunogenic proteins, cell wall degradation/reconstruction enzymes, proteases and esterase, energy metabolism and protein metabolism related proteins, oxidoreductases, and proteins of unknown function.The cell wall degradation/reconstruction enzymes digest the plant cell wall to initiate the invasion. Meanwhile, the elicitor proteins and antigens trigger the immune response of the host plant. Other metabolism related enzymes are responsible for fighting against the oxidative stress and nutrition limitation. Eventually, the pathogens successfully infected the host plants.Moreover, we overexpressed the Yeast homolog Npc2in Escherichia coli and purified the recombinant protein by using the Ni-NTA affinity chromatography and size exclusion. After the crystal screening and optimization, we collected a set of diffraction data at3.3A resolutions.In addition, we improved the activity of Bacillus thuringiensis CrylAa and CrylAa by site-directed mutagenesis. The higher activity of mutants CrylAa H168R, CrylAa N372G and CrylAa N372A have been confirmed by a simple procedure based on the cultured insect cells.