Studies On The Evolution Of Fusarium Oxysporum F.sp. Cubense Strains And Identification And Functional Analyses Of Pathogenicity Related Effectors From Foc TR4

Banana is one of the most important fruits in the world, and it is also the fourth largest food crop after rice, wheat and corn, so the banana production is critical to the world food security, regional development and human health. However, the large-scale outbreak and epidemic of Fusarium oxysporum f.sp. cubense tropical race 4（Foc TR4） has seriously threatened the sustainable development of the industry. In order to prevent and control the disease, we have re-sequenced 56 Foc isolates（covering 4 races and 24 VCGs） genomes, studies on the evolution of worldwide Foc and identified and functional analysised the effectors from Foc TR4 using comparative and functional genomics. The detailed results are as follows.1 Genome re-sequencing and bioinformatics analysis of Foc（1） Genome re-sequencing and assembly55 Foc isolates（covering four races, 24 VCGs） from different banana producing countries had been re-sequenced, including 6 strains of Foc TR4, 8 Foc STR4 isolates, 35 isolates of Foc race 1 and 2 strains of Foc race 2, 2 Foc strains from Dajiao, 2 nonpathogenic isolates of Fusarium oxysporum, and the coverage is above 50 folds. The filtered clean reads were assembled into Contigs and Supercontigs.（2） Differentiation of the Core and LS-Region of the genomeCombining the published genome sequence of 14 Fusarium strains on the Broad Institute website, we identified core regions and Lineage-specific regions（LS regions）of the Foc TR4 strains ii5. Using it as the reference, we found the core regions and LS regions in all the sequenced strains by the method of eliminating the core genome. The results showed that there are significant differences existing in different types of strains on Core and LS region size.（3） Evolution principles of worldwide FocUsing three methods, we investigated the evolution principle of Foc worldwide（Phylogenetic tree using 10 conservative genes, Phylogenetic tree basing on genome-wide SNP and Principal component analysis result, PCA） and found that the genomes of Foc TR4 strains are very conservative, is monophyly and its evolution speed is very fast, while the evolution of Foc STR4 and race 1 is more complex, and they have multiple evolutionary origins, especially the Dajiao isolate, which evoluted from Race 1 and overcome the immune system of Dajiao banana（which in the past, is highly resistant to Race 1, Foc STR4 and Foc TR4）.（4） Determining of the mating-type of FocCollecting of mating type locus sequences of mat1-1 and mat1-2 as the reference, we mapped the assembly sequence onto it and got the mating type genes of all the isolates. The results showed that Foc is the heterothallic fungi, and Foc TR4（VCG01213, 01216 and 01213/16）belongs to mat1-1.（5） Prediction of the effector genes of FocAccording to the structural characteristics of the effector proteins, we predicted the effector genes from each sequenced strains. Combining the published genome sequence of 14 Fusarium strains on the Broad Institute websites, we identified the specific effector genes for each VCG and Race using comparative genomic approaches.2 Expression of the effector proteins during Foc TR4 invasionThe infection process can be divided into four stages, including attachment, symbiosis, and transition from symbiosis into saprophytic and saprophytic. The functions of effector proteins are focus on:（1）a large number of effector proteins is plant cell wall degrading enzymes, such as pectate lyase, pectinesterase, glycosyl hydrolases, cutinase andcellulose.（2）Protect their cell wall by binding a chitin-binding protein.（3） Cell autophage related proteins which are constitutive highly expressing during the whole infecting process. This gene maybe involved in morphogenesis and general pathogenicity.（4） Involving in endocytosis.（5） Clean the ROS which was produced by the host.（6） unknown functions.3 Functional analyses of some important effector proteins of Foc TR4107 genes were selected from the previous proteomic data and the identified effector genes by comparative genomic approach for prokaryotic expression. The expressed proteins were inoculated onto banana leaves, and 20 genes were screened of causing HR response.The incidence of mutants of FOIG₀4525,07508,15400,15834 is around 50%,have medium virulence. Some genes which were highly expressing during the invasion period, or producing severe necrotic spots on banana leaves, were knocked out. The results showed that mutants of FOIG₀0012, 03734 and 07201 lost virulence completely. Knockout mutants of FOIG₀1651, 03512, 03690, 04451,07803,10415,01077 and 01919 only had very weak virulence. This suggested that many effector proteins have important influence on the pathogen pathogenicity.4 Beauvericin is an important virulence factor for Foc TR4Our previous studies have shown that Beauvericin（BEA） could rot pesodostem, kill the suspension cells, kill and decay banana seedlings. And the RNASeq data showed that BEA inhibited DNA replication of the host. In order to further determine the effects of BEA on the pathogenicity of pathogens, we knocked out Bbeas gene, the rate limiting enzyme of BEA biosynthesis. Both of the BEA biosynthesis and pathogenicity in the mutants decreased, comparing to its wild type, while the revertant strain of Bbeas-complement could restore the BEA biosynthesis and pathogenicity, which suggested that the BEA is one of the key factors for pathogen.