Comparative Genome And Transcriptome Analysis Of The Tangerine Pathotype Of Alternaria Alternata And Functional Analysis Of Genes Involved In The Central Regulatory Pathway Of Conidiation And The High Osmolarity Glycerol Pathway In Penicillium Digitatum

Part One:Comparative genome and transcriptome analysis of the tangerine pathotype of A.alternataAlternaria brown spot (ABS) is a highly destructive disease of tangerines grapefruit, and hybrids of grapefruit and tangerine, and tangerine and sweet orange. Severely infected leaves and fruits may drop, and entire shoots may wilt and die, causing significant losses both in terms of yield and marketability of susceptible citrus cultivars. The tangerine pathotype of A.alternata(or A. alternata pv. citri) producing the unique host-selective ACT toxin is the causal agent of citrus brown spot. Both the production of ACT toxin and the ability to detoxify reactive oxygen species (ROS) are required for A. alternata pathogenicity in citrus. However, knowledge about the process of ACT toxin biosynthesis and the underlying regulatory networks of this fungus to cope with ROS is far from clear. In addition, little is known about some basic biological processes, such as response to adverse situations, the sporulation and secondary metabolite biosynthesis. To develop new strategies for effectively controling ABS, deep insights into pathogenesis, development and reproduction of the tangerine pathotype of A.alternata are absolutely required. A well-presented genome will offer great facility to carry out researches regarding those aspects. Therefore,we performed the whole genome sequencing of the tangerine pathotype strain Z7 of A. alternata and the transcriptome sequencing of this fungus under the H2O2 treatment. The information we got is reported as following:1. Comparative genome analysis revealed the unique genes in the tangerine pathotype of A.alternata.The genome assembly of A.alternata Z7 included 161 contigs with a total genome size of 34.41Mb. The average G+C content is 51.0%. The genome encodes 12062 proteins and 115 tRNAs; the average gene length is 1726bp and repeat sequences compose 0.55% of the genome. After comparing orthologous groups of different A.alternata species,10 unique genes were finally found in the genome of the tangerine pathotype; these genes were clustered in the genome and subsequently conformed to be crucial components of the ACT toxin synthesis cluster and are concluded to be the only determinant factor for the pathogenic specialization. A phylogenetic tree constructed based on a combined analysis of 200 single-copy orthologs randomly selected from 26 Alternaria species and other 4 phytopathogenic fungi concides with the classification concept of section Alternaria, section Porri and Section Brassicicola put forward by Lawrence and provide powerful supports for the new proposed taxonomy system in genus Alternaria. A total of 18 secondary metabolite biosynthetic gene clusters or partial clusters were predicted in Z7, the host selective ACT-toxin cluster was found to be 91.2 kb in length containing 25 genes and most of them cotained 2-3 copies, indicating the strong ability Z7 may have to synthetize ACT toxins. An extensive search of the carbohydrate-active enzymes suggested that Z7 possess a great many cell-wall degrading enzymes, consistent with the necrotrophic lifestyle of this fungus.2. Comparative genome analysis revealed the origin of CDC may be vertically transmitted from ancestor of Alternaria.A total length of 1.88Mb of CDC was predicted. Genome structure of CDC is quite different from that of EC. The average G+C content of CDC was calculated to be 47.7%, the repeats rate is 1.23%. The Z7 CDC was predicted to comprise 525protein coding genes, with a number of genes function unknown, and no tRNA was found in CDC. Function analysis revealed enrichment of genes involved in metabolic process and oxidation reduction reactions in CDC. Only a few genes belonging to CAZymes, secretome, kinases and transporters were identified in CDC, Variation of codon usage adaption between EC and CDC encoded genes revealed different evolutions between CDC genes and those resided on essential chromosome (EC). Ka/Ks analysis suggested that CDC are under strong purifying selection. Distribution of orthologous genes among Alternaria species reflected that nearly 90 percent of the CDC genes have more than 5 clear orthologues in the other Alternaria species and over 95% of CDC proteins showed strong similarities to proteins from the other Alternaria species, supports the notion that the origin of Z7 CDC may be vertically transmitted from ancestor of Alternaria.3. Comparative analysis of mitochondrial genome revealed the great disparities in terms of gene arrangement.The whole mitochondrial genome of A.alternata was also obtained and annotated. Z7 mtDNA was 50,625bp in length with average A+T content of 70.8%; it encodes 13 standard proteins,2 ribosome subunits and 31 tRNAs; the coding efficiency of the whole mitochondrial genome is 63.7% and shows priority to utilize codons rich in A/T. The comparison among different species in Pleosporales showed that sequences for most mitochondrial genes are highly conserved, however, the arrangement for them displayed wide differences, indicating dinstinct evolutionary history. Phylogenetic analyses based on 12 concatenated mitochondrial proteins indicated that A.alternata clustered with members of the order Pleosporales, which is in agreement with previous results. Moreover, the introns and intergenic regions in mitochondrial genome varied across species, which may be the main factor for the different mitochondrial genome sizes.4. Comparative transcriptomics revealed that the glutathione system, peroxidases and protein families like transporters play an important role in elimination of ROS in A. alternata.Comparative transcriptome analysis was performed to determine gene expression changes after H2O2 treatments for 30 min.1108 genes were upregulated while 498 ones were downregulated. GO analysis revealed enrichment of these differentially expressed genes involved in cellular metabolic process, oxidation reduction and transmembrane transport. The glutathione system, thioredoxins, peroxidases are the main scavengers in eliminating H2O2. Further investigation into protein families of kinases, transcription factors, cytochrome P450, ubiquitin and heat shock proteins helped to identify and obtain many genes associated with adaptation to oxidative stress; also 29 genes located in CDC were found to be induced in the condition of H2O2, including the polyketide synthetase CDCn|11750 which plays essential role in ACT toxin biosynthesis.Part Two:Functional analysis of genes involved in the central regulatory pathway of conidiation and the high osmolarity glycerol pathway in Penicillium digitatum1. PdbrlA, PdabaA and PdwetA control distinct stages of conidiogenesis in Penicillium digitatumReproduction of massive asexual spores is the primary factor that contributes to the epidemic of citrus green mold. To understand the molecular mechanisms underlying conidiogenesis in P. digitatum, we functionally characterized the Aspergillus nidulans orthologs of br1A, abaA and wetA. We showed that deletion of PdbrlA completely blocked the formation of conidiophores, whereas deletion of PdabaA led to the formation of aberrant and non-functional phialides. The PdwetA mutant showed various defective phenotypes, such as abnormal conidia with loose cell wall, delayed germination and reduced tolerance to stresses of osmotic, detergent, heat shock and menadione but elevated resistance to H2O2. Pdbr1A influenced genes were identified by comparing the global gene expression profiles between the wild-type and the PdbrlA deletion mutant during conidiation. Gene ontology analysis on these differential expressed genes (DEGs) revealed the diverse roles of Pdbr1A in metabolism, transportation and cell structure. Moreover, out of 39 genes that previously reported to be involved in conidiogenesis in Aspergillus, mRNA levels of 12 and 2 genes were depressed and induced, respectively, in the APdbr1A. All the 12 downregulated genes have putative Br1A or AbaA response elements.6. Os2 MAP kinase-mediated osmostress tolerance in Penicillium digitatum is associated with its positive regulation on glycerol synthesis and negative regulation on ergosterol synthesisThe high osmolarity glycerol (HOG) pathway is ubiquitously distributed among eukaryotic organisms and plays an important role in adaptation to changes in the environment. The Hog1 ortholog in Penicillium digitatum, designated Pdos2, was identified and characterized using a gene knock-out strategy. The â–³Pdos2 mutant showed a considerably increased sensitivity to salt stress and cell wall-disturbing agents and a slightly increased resistance to fungicides iprodione and fludioxonil, indicating that Pdos2 is involved in response to hyperosmotic stress, regulation of cell wall integrity and sensitivity to fungicides iprodione and fludioxonil. Surprisingly, the mutant was not affected in the response to oxidative stress caused by H2O2. The average lesion size in citrus fruits caused by â–³Pdos2 mutant was smaller (approximately 25.0% reduction) than that caused by the wild-type strain of P. digitatum at 4 days post inoculation, which suggests that Pdos2 is needed for full virulence of P. digitatum. Interestingly, in the presence of 0.7M NaCl, the glycerol content was remarkably increased and the ergosterol was decreased in mycelia of the wide-type P. digitatum, whereas the glycerol content was only slightly increased and the ergosterol content remained stable in the â–³Pdos2 mutant, suggesting that Pdos2-mediated osmotic adaption is associated with its positive regulation on glycerol synthesis and negative regulation on ergosterol synthesis.