Function Analysis Of Pathogenic Related Gene VDMSB In Verticillium Wilt And Genome-Wide Identification Of Chitin Related Gene Family In Cotton

Cotton （Gossypium spp.） is the most important source of natural textile fibers worldwide, and is also a significant oilseed crop. Verticillium wilt severely damages cotton production, V. dahliae can cause such severe yield and quality losses in cotton that considered as a serious disease of host plants. Study on pathogenic related gene in V.dahliae and resistant gene in cotton, not only provide the basis of cotton-V. dahliae interaction mechanism, but also providing an important candidate gene for cotton breeding.Recent research suggests that MAPK （Mitogen-activated protein kinases） signal pathway plays an important role in plant pathogenic, such as:fungal hyphae growth, adhesion on roots of the plant, special invasion growth structure and so on. Several MAPK genes have been identified from a variety of plant pathogenic, including the Msb2 gene which located in the upstream of the MAPK pathway and play an important role in pathogen infect the host plants. In nature, plants activate the complex immune response and resistance to fungal infection by perception of the PAMPs （Pathogen associated molecular patterns）. Among them, the chitin is a typical PAMPs, which has been considered to be an important component of the cell walls of fungi. The previous studies have shown that there are two categories chitin related genes in plant:chitin receptor proteins and chitinase. Chitinase degrade the cell walls of fungi and release small molecule chitin oligosaccharide, thus inhibiting the growth of fungi. And chitin receptor protein can specifically identify the chitin oligosaccharide and activate the plant immune responses by a series of signal pathway. Therefore, these two kinds of chitin related genes play an important role in the immune response of plants.In this study, the defoliating type strain Vd8 was using as material, we cloned the VdMsb gene and studied on its function; the genome data subsets of cotton A and D subgroups have been released, performed a genome-wide analysis, classification and function analysis of LysM gene family in cotton. The main results are as follows:1. In this work, We cloned a transmembrane mucin protein, VdMsb, using the homology cloning strategy. VdMsb contained an ORF of 2688 bp without introns that encoded an 895 residue protein （GenBank No:KM032761） with a molecular mass and pI of 90.6 kD and 4.33, respectively. The deduced VdMsb protein contains a conserved signal sequence, a transmembrane region and a highly conserved cytoplasmic tail, and the amino acid sequence shared 49.27% and 48.78% identity with homologs in F. oxysporum Msb and M. oryzae Msb, respectively. Alignment with homologs from other fungi revealed highly conserved C-terminal and transmembrane regions.The gene deletion and complement mutants were obtained by ATMT method. To analyze the function of VdMsb in fungal growth and microsclerotia production, gene deletion mutant Amsb and gene-complemented strain Amsb+Msb were grown on CM medium and compared with the wild-type strain Vd8. The colony morphology in the wild-type and complemented strains were very similar with production of microsclerotia, while the mutant was basically light color showing that microsclerotia development was clearly affected. The results indicated that VdMsb played a positive regulatory role in microsclerotia production in V. dahliae. To further investigate the role of VdMsb in plant infection, both cotton and Arabidopsis seedlings were inoculated with a spore suspension of the V. dahliae deletion and complement mutants, with the wild-type Vd8 as control strain. The disease index was 100% for both inoculated with wild-type Vd8 and Amsb+Msb strains, but only 25% for the negative control and 37.5% for the Amsb strains, respectively. Similar results were obtained from virulence assays on Arabidopsis thaliana. The plant inoculated with the wild-type strain showed a progressive increase in disease symptoms, whereas inoculated with the Amsb strains showed reduced but still produced a low level of disease symptoms. These results strongly indicated that VdMsb is essential for the pathogenicity of V. dahliae.Further analysis found that, the spore concentration of the Amsb mutant （2.63±0.86 ×107） was significantly less than that of the wild-type strain （4.49±1.01×107） and the Amsb+Msb strain （4.82±0.97×107）, which were comparable. To assess adhesion capacity, Arabidopsis thaliana roots were immersed in suspensions containing spores from the different mutants, incubated for 36 h, and analyzed by scanning electron microscopy. It was observed that root colonization was significantly diminished in the Amsb mutant as compared with wild-type and the complemented strain. To investigate the diminished pathogenicity of the Vdmsb deletion mutant, cotton roots were inoculated with microconidia from the different mutants and the phenotype was assessed three days after inoculation using a stereoscope. The stem vascular bundles of plants inoculated with wild-type Vd8 and the Δmsb+Msb complemented strain were brown in color, indicating that pathogen has invaded into the vascular bundles and spreading. While the color of vascular bundles in plants infected with the Δmsb mutant was similar to control plants. These results indicated that, the Vdmsb gene is essential for the spore production, invasion growth and adhesion capacity of V. dahliae.2. To clarify the distribution of LysM gene family in two cotton subgroups,28 and 26 members were identified from Graimondii and G. arboreum genome, respectively. The gene family was divided into four categories according to the characteristics of the domain, included the LYK, LYP, LysMe and LysMn. The chromosome distribution result showed that the LysM genes matched to 12 chromosomes, except D7 and were designated GrLYK1-GrLYKS, GrLYP1-GrLYP4, GrLysMel-GrLysMe7 and GrLysMnl-GrLysMn7, respectively, accorded by the location on chromosomes. The LysM genes of A subgroup were named based on the homologs with D subgroup.We analyzed the expression pattern of 12 LysM gene in Hai7124 tissues using the qRT-PCR. There were four genes highly expressed in all kinds of tissues, included GbLYPl, GbLYP2, GbLysMn3 and GbLysMn7. The GbLYK1, GbLYK5, GbLYK7, GbLysMel and GbLysMe2 were highly expressed in root tissues. GbLYP4, GbLYK8 and GbtYPS were highly expressed in stem and leaf tissues.The results showed that, there are three genes high expression in the root:GbLYP1, GbLYK5 and GbLysMn7. Among them, the expression of GbLYPl and GbLYK5 were significantly increased after V. dahliae induced.We cloned two resistant related gene, GbLYP1 and GbLYK5, by homology cloning strategy. GbLYPl contained an ORF of 1080 bp that encoded an 359 residue protein with a molecular mass and pI of 38.7 kD and 5.54, respectively. GbLYK5 contained an ORF of 2013 bp that encoded an 670 residue protein with a molecular mass and pI of 73.7 kD and 5.90, respectively.The function analyzed of candidate genes were performed by VIGS. The cotton seedling appeared typical sense of verticillium wilt disease symptoms, included wilting and fall off, which inoculated with plRV2-GbLYK5 and pTRV2-GbLYPl. The disease index were reached to 73% and 66.7%, respectively, which were significantly difference with the control group,35.4%. These results strongly indicated that GbLYP1 and GbLYKS may positively regulate the process of cotton resistant the V. dahliae.3. We identified 49 and 46 members from Graimondii and G. arboreum genome, respectively. The gene family was divided into five categories according to the characteristics of the domain, included Class I to Class V. The chromosome distribution result showed that the Chitinase genes matched to 12 chromosomes except D4 and were designated GrChil-GrChi46 accorded by the location on chromosomes. The chitinase genes of A subgroup were named based on the homologs with D subgroup.We analyzed the expression pattern of 37 chitinase gene in Hai7124 tissues using the qRT-PCR. The results showed that, there were two genes highly expressed in all kinds of tissues, included GbChi31, GbChi46; There were 21 genes highly expressed in root included GbChi3 and GbChi22, which revealed the highest expression levels. There were 7 and 9 members highly expression in stem and leaf, respectively. Fungal-induced expression analysis indicated that the expression levels of GbChi3, GbChi22, GbChi31 and GbChi46 significantly increased, which GbChi22 was significantly increased highest expression level after V. dahliae induced.We cloned one resistant related gene, GbChi22, by homology cloning strategy. GbChi22 contained an ORF of 1107 bp that encoded an 368 residue protein with a molecular mass and pI of 93.6 kD and 5.04, respectively. The deduced protein contains a signal sequence, one glycol_hydro₁8 domain.The function analyzed of candidate genes were performed by VIGS. The cotton seedling appeared typical sense of verticillium wilt disease symptoms, included wilting and fall off, which inoculated with pTRV2-GbChi22. The disease index was reached to 68.8%, which was significantly difference with the control group,35.4%. These results strongly indicated that GbChi22 may positively regulate the process of cotton resistant the V. dahliae.