| Curvularia leaf spot mainly caused by Curvularia lunata(Wakker) Boed. is widely distributed in the world. The pathogenicity differentiation of this pathogen easily takes place, which usually causes the resistance lose of cultivars to the pathogen. In this study, resistant maize cultivars were used to induce the pathogenicity variation of C. lunata. Then, the dynamic progress of the pathogenic differentiation were studied, the sensitive degree of the pathogen to selective pressure of resistant hosts were analyzed, and pathogenicity variation-associated proteins and genes were screened to reveal the molecular basis of virulence differentiation, which would have practical and theoretical values in warning and forecasting this variation caused by the unreasonable use of resistant cultivars containing tropical and subtropical blood relationship.This research mainly focused on the genome sequence of C. lunata, the analyses of genome sequences, the impact of host selective pressure of maize germplasm with different resistance on pathogen pathogenicity, the gene expression difference between strain with different virulence by proteomics and transcriptomics, the function of gene related to pathogenicity by genetics. The main research contents and results are shown as the followings.1. Genome sequencing and sequences analyses. Firstly, the full genome of C. lunata CX-3 was sequenced, and sequenced data was jointed and assembled. Secondly, gene prediction and gene family classification were conducted, and genes related to pathogen-host interaction(PHI) were screened. Lastly, comparative genome analyses were performed. The main results are as the followings.(1) The assembled size of C. lunata CX-3 genome is 35.5 Mb containing 11234 gene-encoded proteins, 840 of which were secreted proteins.(2) The C. lunata CX-3 genome has 2830 conserved protein families, which contain 8471 proteins including 161 transposases, 129 G-protein-coupled receptors, 153 protein kinases, 76 small secreted cystein-rich proteins(less than 150 AA), 235 glucoside hydrolases, 47 ABC transporters, 252 MFS transporters, 146 P450 s, 36 backbone genes required for the biosynthesis of secondary metabolites.(3) 1904 genes related to the pathogen-host interaction were identified including 127 protein kinases, 70 glucoside hydrolases, 40 ABC transporters, 167 MFS transporters and 112 P450 s, etc.(4) Protein sequences of C. lunata CX-3 showed 85.9%, 78.6%, 75.8%, 70.9% and 47.9% identities with C. lunata m118, B. maydis C5, S. turcica, P. tritici-repentis and M. oryzae, respectively. A phylogenomic analysis reveals that C. lunata was diverged from B. maydis around Cretaceous Extinction(65 million years ago).(5) The C. lunata CX-3 has high frequencies of Câ†'G and Tâ†'A mutation bias, which is an important genetic proof for explaining the virulence differentiation of C. lunata. In a word, the genome information of C. lunata CX-3 will lay the basis for fully revealing molecular mechanisms of pathogenicity and virulence differentiation of C. lunata.2. Impact of the selective pressure of host on the pathogenicity. Firstly, C. lunata strain WS18 with weak virulence was subinoculated on two resistant maize cultivars(Pob21 and Pob101) for 11 generations and one susceptible maize cultivar(HZ4) for 6 generations to study the impact of the selective pressure of host on the virulence. Then, the genetic stability of the pathogenicity variation was analyzed by removing the host selection pressure from strains. The main results are as the followings.(1) Resistant cultivars significantly induced the virulence increase of pathogen.(2) Susceptible cultivar did not obviously induce the pathogenicity changes.(3) The degree of virulence increase induced by resistant cultivars was significantly weakened after removing the selection pressures of resistant cultivar. These results show that the induction of resistant host is a potential way by which the adaptive pathogencity variations of pathogens take place, and host selective pressures increase the risk of resistance loss of cultivars.3. Screening of differential proteins related to virulence increase. Firstly, the experiment conditions for 2DE were optimized. Secondly, 2DE was conducted for proteins of the 0th, 5th, 8th and 11 th strains induced by Pob21 to screen differential proteins. Lastly, differential proteins were identified by MALDI-TOF-MS/MS. The main results are as the followings.(1) p H5-8 is the optimal p H value for IEF.(2) 16 differential proteins with 1.5 fold changes were screened in the 11 th induced strain(WS18-Pob21-11), of which 12 were up-regulated such as melanin biosynthesis-associated proteins Brn1, Brn2 and stress related protein HSP 70, and 4 were down-regulated such as SCD being involved in melanin biosynthesis.(3) The translational levels of Brn1, Brn2 and HSP70 were positive correlation with virulence among the 0th,5th, 8th and 11 th strains.4. Screening of differential genes related to virulence increase by constructing a suppression subtracted hybridization(SSH) c DNA library. Firstly, c DNA of the 0th(WS18) and 11th(WS18-Pob21-11) induced strains were served as driver and tester respectively to construct a SSH library. Secondly, positive clones with single insertion sequence were screened, and the sequences were assembled to unigenes after removing linker and vector sequences. Lastly, differential genes were analyzed with bioinformatics and confirmed by RT-q PCR. The main results are as the followings.(1) 188 differential unigenes were obtained including 158 known genes and 30 novel genes.(2) 14 of 158 known genes were involved in pathogenic process such as brn1, ubiquitin encoding gene, laccase-1 precursor encoding gene and ace1, and stress response such as sod, tsa1 and hsp104, which were close to virulence increase.5. Screening of differential genes related to virulence increase by RNA-seq. Firstly, the transcriptome sequencing of the 0th and 11 th induced strains was performed by RNA-seq. Then, differential genes were analyzed using bioinformatics. The main results are as the followings.(1) 200 and 164 differential genes are up-regulated and down-regulated in the 11 th induced strain, respectively, compared to the 0th induced stain, of which 359 are known genes.(2) The up-regulated genes are mainly rich in transport, metabolic process, mycelium development, response to stress, pigment biosynthetic process and proteolysis, etc.6. Comprehensive analyses of proteins and genes related to the adaptive virulence increase. Proteins and genes related to the virulence increase were screened by comparative analyses of differential proteins and genes from 2DE, SSH library and RNA-seq combined with their biological functions, and the regulation network of pathogen virulence increase was drawn. The main results are as the followings: 33 proteins and genes related to virulence increase were obtained including 4 melanin biosynthesis-associated proteins and genes, 14 genes being located in a toxin biosynthesis-associated gene cluster, 4 stress resistance proteins and genes, 4 multi-drug transporter-encoding genes, 2 transcript factors and 5 other genes related to pathogenicity. The results show that the regulation networks of melanin, toxin, stress resistance and cellulase are involved in the adaptive virulence increase, and there are connections among the melanin, toxin and stress resistance regulation networks; sod, brn1, scd, PKS, ace1, tsa1, hsp7 and hsp104 are probably related to the adaptive virulence increase and served as candidate marker genes of the adaptive virulence increase.7. The expression analysis of virulence increase-associated marker genes(sod, brn1 and scd) in different pathogenic types of strains. Firstly, the pathogenic types of 5 C. lunata strains were identified using eight differential hosts. Then RT-q PCR was performed to study the corresponding relation of the transcriptional levels of sod, scd, brn1, tsa1, pks and hsp 70 genes and different pathogenic types. The main results are as the followings.(1) The transcriptional levels of sod and tsa1 gene in high and moderate pathogenic types of stains were more than that in low and special pathogenic types of strains.(2) The transcriptional levels of brn1, scd, pks and hsp70 were not obviously corresponding to pathogenic types. These results showed that the transcriptional level of sod and tsa1 gene was corresponding to pathogenic types of pathogen, and sod and tsa1 could be used as a marker gene of virulence differentiation.8. Function analysis of sod gene. Firstly, deleted mutant and complementary mutant of sod gene were constructed, respectively. Secondly, biological characteristics of the wild type strain, Δsod, and complement mutant were compared. Lastly, RT-q PCR was performed to analyze the effect of sod deletion on the expression of two melanin biosynthesis associated genes(brn1, scd) and a toxin biosynthesis associated gene(clt-1). The main results are as the followings.(1) There were no distinct differences in colonial morphology, CWDE activities and toxin virulence among the three strains.(2) Δsod melanin production was significantly reduced compared to the wild type strain and complement mutant, cultured in PD medium for 48 h.(3) The virulence of Δsod was lower than the wild type strain and complement mutant at early stage of infection, but its virulence returned to the similar level of the wild type strain and complementary mutant at late stage of infection.(4) The deletion of sod did not significantly effect the expression of brn1, scd and clt-1. These results show that sod gene contributes to the pathogen pathogenicity, but it is not essential for pathogenicity; sod influences the production of melanin, but how the influence work is not clear.In conclusion, the genome information of C. lunata CX-3 laid the basis for fully revealing the molecular mechanism of pathogenicity and virulence differentiation. The selective pressures of resistant hosts were able to induce the virulence increase of pathogen, in which the regulation networks of melanin, toxin, stress resistance and cellulase were involved. Marker genes related to virulence increase could reflect the virulence differentiation of pathogens in the translational or transcriptional levels. sod and tsa1 as a marker gene of virulence differentiation contributed to pathogenicity of C. lunata in the early stage of infection by influencing melanin production. |
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