| Rubber tree (Hevea brasiliensis Musll-Arg.), perennial crop that has found its place in many national economies of producing countries. It is of major economic importance because the milky latex extracted from the tree is the primary source of natural rubber that is an important industrial raw material as well as strategic and critical material that contributes to the welfare of local small farmers. Colletotrichum gloeosporioides is a pathogen in rubber trees causing huge yield losses. At present, the prevention of Colletotrichum leaf disease in the rubber tree is largely relied on the use of chemical fungicides. However chemical disease control might cause problems ranging from pathogen resistance to chemical agents, the risk of poisoning humans or animals, harm to natural enemy, to the contamination of livestock products, food products, atmospheres, waters and soil. There is still little known about the pathogenesis of C. gloeosporioides, and the molecular interactions between this pathogen and rubber tree. To further elucidate the disease occurrence law, especially molecular mechanisms of C. gloeosporioides pathogenicity, breeding resistant rubber tree clones, innovating control strategy, are being addressed for sustainable development of the natural rubber industry.The study focused on these aspects. One strain (HBCg01) with high virulence was selected from our large collections of C. gloeosporioides isolated from rubber growing regions in China. Its genome sequencing project was accomplished by our laboratory with the joint efforts of Beijing Genomics Institute (BGI). A T-DNA random insertion library of C. gloeosporioides transformants (HBCg01) was generated thought Agrobacterium tumefaciens-mediated transformation (ATMT). Bioassays were established for (?) evaluating resistance of 46 rubber tree clones to Colletotrichum gloeosporioides; (?) large-scale screening of the pathogenicity mutants and pathogenicity assays. Then the resistances of 46 rubber tree clones to Colletotrichum gloeosporioides were evaluated. All of T-DNA insertion transformants of C. gloeosporioides were screened for mutants defective in pathogenicity, which were subjected to morphological observation and molecular analysis subsequently. A thermal asymmetric interlaced PCR (TAIL-PCR) protocol was used for cloning genomic DNA flanking inserted T-DNA. Genomic sequences flanking T-DNA were recovered from mutants by TAIL-PCR, and were used to isolate the tagged genes from the genome sequence of wild-type C.gloeosporioides by Basic Local Alignment Search Tool searches against the local genome database of the wild-type C. gloeosporioides. The FGENESH program (Softberry Inc., Mount Kisco, NY. U.S.A.) was used for gene structure prediction using the Alternaria, Botrytis, Fusarium graminearum and Magnaporthe oryzae etc. matrices. Using appropriate BLAST algorithms, sequence homology searches were performed against the NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) nr protein database. Meanwhile the function of one putative gene identified in our screen, designated as CgATPase was analyzed by gene knockout/complementation. The specific results were as following:The whole genome of C. gloeosporioides (HBCg01) were paired-end sequenced using high-throughput Illumina sequencing technologies, constructed the insert fragment 500 bp library, the expected data volume 2500 Mb. Whole genome was sequenced and data were assembled. The total length of the whole genome is 55.495 Mb with 53.62% of GC percentage,15-mer expect to a depth of 34. Non-consensus sequence was found. This indicated that the sequencing data could be fine map sequencing and analysis.A high quality ATMT library of 4,128 C. gloeosporioides transformants was generated. Southern blot analysis showed 60.4% of the transformants had single-site T-DNA integrations. The transformation efficiency was up to 150-400 transformants per 106 conidia. Positive transformants accounted for 86.96% of the total by PCR assay. All of the transformants tested remained mitotically stable, maintaining their Chlorimuron-ethyl resistance after several generations of growth in the absence of Chlorimuron-ethyl. The GFP signal could be detected in conidia and hyphae of tested transformants. And the gfp gene could be detected by PCR, confirming stable maintenance of the marker gene across successive generations.Three methods to identify resistance/susceptibility to CLD of 46 rubber clones were examined in this study. The resistance was divided into 5 grades:highly resistant (HR), resistant (R), moderately susceptible (MS), susceptible (S), and highly susceptible (HS). Analysis of results of resistance/susceptibility of 46 rubber clones through three methods showed that among the 46 rubber clones,0 highly resistant (HR),8 resistant (R),19 moderately susceptible (MS),16 susceptible (S), and 3 highly susceptible (HS), accounted for 0%,17.39%,41.3%,34.78,% and 6.52% respectively. Copper brown leaves were the most susceptible to the pathogen. The results of our research provided important information for disease resistant breeding, clone utilization as well as the management of CLD. Based on experiments, we developed a screening method for pathogenicity of C. gloeosporioides transformants: in a primary screen infection assays were performed on intact, detached copper brown rubber tree leaves by inoculating agar plug cut from mycelial mats, in a secondary screen by inoculation conidial suspensions at least three times.Transformants were screened for defects in pathogenicity with a detached copper brown leaf assay.32 mutants showing reproducible pathogenicity defects were obtained.16 genomic sequences flanking T-DNA were recovered from 15 pathogenicity mutants by thermal asymmetric interlaced PCR, and were used to isolate the tagged genes from the genome sequence of wild-type C. gloeosporioides by Basic Local Alignment Search Tool searches against the local genome database of the wild-type C. gloeosporioides. Two known C. gloeosporioides pathogenicity genes (Mut-24 and Mut-28) were identified in our screen such as those encoding Glomerella cingulata hard-surface induced protein namely Chip5 and C. gloeosporioides regulatory subunit of protein kinase A (PKAR) gene involved in cAMP-dependent PKA signal transduction pathway (E?3e-135). One potential pathogenicity genes (Mut-29) encoded calcium-translocating P-type ATPase containing a conserved domain of P-type ATPase (E= 0).Two putative genes encoded Glycosyltransferase family 28 domain-containing protein (Mut-4) and Mov34/MPN/PAD-1 family protein (Mut-15), respectively (E?1e-171). Six potential pathogenicity genes had no known homology in filamentous fungi and were likely to be novel fungal virulence factors (Mut-7, Mut-9, Mut-10-1, Mut-13, Mut-19, and Mut-23). The rest had Putative function matched (E?1e-159) with putative protein of C. higginsianum/G. graminicola with unknown function (Mut-8, Mut10-2, Mut-18, Mut-26 and Mut-30). Agrobacterium-modiated insertional mutagenesis was used successfully to identify pathogenicity genes in C. gloeosporioides.The function of one potential pathogenic gene was analyzed in detail by gene knockout/complementation technology. Database searches (Blastx) revealed that the rescued sequence has strong amino acid similarity to many calcium p-type ATPase of fungi, the C. Gloeosporioides gene presumably required for pathogenesis toward rubber tree was named the cgATPase gene. P-type ATPases are important enzymes that carry out many fundamental processes in biology and medicine, ranging from the generation of membrane potential to muscle contraction and the removal of toxic ions from cells. However, the biological function of the CgATPase gene for its growth, development and pathogenicity is little known.The loss of CgATPase affects hyphal growth, colony character and sporulation. The ?CgATPase mutant displayed defects in infection of the host plant. While the reintroduced transformant was able to partly restore the pathogenicity of C. Gloeosporioides. Most conidia of ?CgATPase mutant failed to penetrate the onion epidermis to form primary hyphae in the cells of onion epidermis. The wild type strain and CgATPase reintroduced transformant shared very similar biological phemotypes, but different with ACgATPase mutant:(?) the cell wall integrity of ?CgATPase mutant decreased. After treated with different concentrations of SDS, ? CgATPase mutant was severely inhibited on CM media with 0.04% SDS, however wild type strain and CgATPase reintroduced strain was able to grow on CM media with 0.08% SDS. (?) Congo red and Calcoflour white inhibit the growth of ?CgATPas mutant, CgATPase reintroduced transformant as well as wild type C. gloeosporioides, but the inhibiting effect on ACgATPas mutant was higher than that of CgATPase reintroduced transformant as well as wild type C. gloeosporioides. ?CgATPas mutant, CgATPase reintroduced transformant and wild type C. gloeosporioides showed the same level tolerance to salt stress. The growth rate of ?CgATPas mutant, CgATPase reintroduced transformant and wild type strain were not affected by high osmotic stress of sorbitol. CgATPase is not involved in the response to carbendazim and chlorimuron. |
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