Coloning And Functional Identification Of A Mitogen-Activated Protein Kinase Gene(CCK1)and Toxin Ecoding Gene (ct) Of Corynespora Cassiicola-the Pathogen Of Corynespora Leaf Fall Disease Of Hevea Brasiliensis

Hevea rubber (Hevea brasiliensis) was an important tropical crop, and its product was one of the four major industrial raw materials along with steel, petroleum, and coal. Corynespora cassiicola (Bert.&Curt.) Wei, an important plant pathogenic fungus, infected more than300kinds of host plants. The Corynespora Leaf Fall (CLF) caused by C. cassiicola in Hevea rubber was a major disease influencing the production of natural rubber. Currently, foreign and local studies mainly focused on pathogen biology and disease epidemiology as well as control. However, little research had been directed to pathogeneisis mechanisms, especially pathogen-related genes and their regulations. Resaerch on the pathogeneisis-related genes in Hevea-C. corynespora system would be of great academic value in terms of revealing the pathogenesis and understanding the interaction mechanism between C.corynespora and its hosts. Furthermore, the findings would be beneficial to developing new control methods.In this research, full-length sequences of CCKl gene, a MAPK homologous gene, and a toxin gene ct, encoding cassiicolin toxin, were obtained using homology-based cloning method from the genome of C. cassiicola isolated from Hevea rubber. The disrupted genes were designed by insertion mutation with the hygromycin B phosphotransferase gene (hygB), and gene knockout mutants were obtained through protoplast transformation mediated by poly ethylene glycol (PEG), and the phenotypes of disruptants were also determined. The results were mainly as follows:1. According to the conserved amino acid sequence in some filamentous fungal MAPKs, a pair of degenerate primers was designed, and a MAPK homologous gene fragment of PMKl was amplified from the genomic DNA of C. cassiicola. With SEFA-PCR(Self-Formed Adaptor PCR), the5end (1802bp) and3end (652bp) segments flanking the sequences were obtained by two-step amplification. The spliced and corrected DNA was2710bp in size and named CCKl. After comparing the sequences of the CCKl DNA and the cDNA of the homologous gene PMKl, a pair of primers were designed from an external intron. A1065bp cDNA fragment was amplified from the genomic RNA of C. cassiicola by RT-PCR. The initiation codon ATG was found located within1227bp and1229 bp and the neighboring sequence conforms to the Kozak rule, which was the ACCATGT. The stop codon TAA was within2502bp and2504bp. The sequence from ATG to TAA was1278bp, including5exons with110bp,196bp,382bp,348bp, and29bp, respectively (being within the1227-1336,1390-1585,1640-2021,2072-2419, and2476-2504bases), and4introns with56bp,54bp,50bp, and56bp, respectively (being within the1337-1389,1586-1639,2022-2071, and2420-2475bases). The amino acid sequence of the open reading frame was inferred to be354, with a molecular weight of40.98kDa and an isoelectric point of6.43, being a slightly acidic protein. After searching for proteins similar to CCKl in the GenBank database and conducting a phylogenetic tree analysis, CCKl was found to be highly homologous to MAK2from Phaeosphaeria nodorum (XP₀01793869.1), MAPK from Letosphaeria maculans (AAM89501.1), BMK1from Cochliobolus miyabeanus (AB180104.1), CHK1from Cochliobolus heterostrophus (AF178977.1), and PMK1from Magnaporthe grisea, and FMK1from Fusarium oxysporum f.sp. lycopersici and other proteins of the PMKl-type proteins, with similarities of99%,99%,98%,98%,93%, and93%, respectively. Furthermore, a TEY (182AA-184AA) of the MAPK protein activation domain-participating dual phosphorylation and Ser/Thr protein kinase-conserved domain were included. Therefore, the CCKl gene acquired from C. cassiicola in this experiment was an MAPK gene of PMK1type. The sequence had been submitted to GenBank (GU373808.1).2. The pMD18-T vector T cloning site, Sph I, and Sal I enzyme cutting sites were successively inserted into the CCKl gene5end fragment,3end fragment, and hygB gene, thus, successfully constructing the CCKl gene knockout vector pFHB. A recombined CCKl gene fragment from the knockout vector containing the hygB gene was amplified, and used to transform the C. cassiicola protoplast.After PDA agar plate screening, PCR verification, and Southern blot hybridization,the mitogen activated protein kinase gene CCKl knockout mutant was confirmed. Compared with the wild-type strain, the CCKl mutant losed sporulation capacity and the color of the colony changes from brown and gray to blackish green. Aerial mycelial mass significantly reduced, and mycelial growth rate obviously decreased. The hypha was firm and straight with less branches and septa, whereas the puce pigmentation intensifies with evident protoplasm concentration. The mycelium was more capable of withstanding high temperature and the optimal range of environment pH narrowed, whereas osmotic stress response to a variety of salts weakened. The fungus was more sensitive to carbendazim and thiophanate-methyl, as well. Meanwhile, the leaves of the rubber tree inoculated with mycelia plugs did not develop disease but the mutant demonstrates weak virulence when crude toxin was tested. Laccase production was relatively postponed with a relative increase in production. Cellulase production was significantly increased. The growth promoting effects of exogenous Ca2+and the growth inhibiting effects of exogenous cAMP on mycelial growth of the CCK1mutant were significantly higher than that of the wide type. All of the above demonstrated that the mitogen activated protein kinase CCK1might be play a vital regulatory role in mycelial growth, development, cell differentiation, and pigmentation, as well as conidium formation, toxin, laccaes and cellulase production, adaptability to the environment, and pathogenicity of C. cassiicola in Hevea rubber, and might be involve in mycelial growth together with Ca2+or cAMP signaling pathway.3. According to the cas gene sequence (EF667973.1) in NCBI, a pair of degenerate primers was designed, and a toxin encoding ct gene was amplified from the genomic DNA of C. cassiicola by the same method as the CCK1gene. The spliced and corrected DNA was2800bp in size and contained the entire180bp open reading frame. The initiation codon ATG was found located within802bp and804bp and the stop codon TAA was within1095bp and1097bp. The sequence from ATG to TAA was296bp, including3exons with105bp,60bp, and15bp, respectively (being within the802-906,974-1033, and1083-1097bases), and2introns with67bp and49bp, respectively (being within the907-973and1034-1082bases). The amino acid sequence of the open reading frame encoding was inferred to be59, with a molecular weight of5.92kDa and an isoelectric point of5.70, being an acidic protein. After searching for proteins similar to CT in the GenBank database, CT was found to be hightly homologous with pro-cassiicolin from Corynespora cassiicola (ABV25895.1) with similarities of85%. Therefore, the ct gene acquired from C. cassiicola in this experiment might be a toxin encoding gene. The sequence had been submitted to GenBank (GU373809.1).4. The pMD18-T simple vector T cloning site were successively inserted into the toxin encoding ct gene fragment, and then the hygB gene were inserted in antisense orientation into the ct sequence to the engineened Sal â…  site, thus, successfully constructing the ct gene knockout vector pCTH. A recombined ct gene fragment from the knockout vector inserted with the hygB gene was amplified, and used to transform C. cassiicola protoplast by the PEG method. After PDA agar plate screening, PCR verification, and Southern blot hybridization,the toxin encoding ct knockout mutant was confirmed. There were no obvious difference in the colony color, mycelia morphology and growth, sporulation quantity, conidial germination, requirement of temperature and pH, and laccase and cellulase production between the mutant and the wild type. Meanwhile, the leaves of the rubber tree inoculated with mycelia plugs can cause typical symptoms but demonstrate weak virulence. However, when crude toxin was tested for pathogenicity, it displayed weaker virulence. All of the above demonstrated that the toxin was a impotant virulence factor but not a unique factor in the pathogenicity of C. cassiicola in Hevea rubber.