Two Tales Of Penicillium Digitatum: Comparative Genomics And DMI Fungicides Resistance

China is one of the largest Citrus growing and consuming countries in the world, with an annual production more than10million tons. Citrus green mold (CGM) is a long-term issue for fruit storage, transportation and marketing worldwide. In China, CGM causes an annual loss of more than one million tons of Citrus fruit. Currently, it is still challenging to control CGM in the field due to the emergence of fungicide resistant populations, which poses a great threat to the agriculture safety. Thus, deciphering the interaction between host and pathogen, and elucidating the funigicides resistance in field populations are the important tasks for Citrus postharvest management. To pursuit these goals, the whole genome of Penicillium digitatum was sequenced and compared. The genome information, in return, helped to investigate the DMI fungicides resistant populations collected in China. The major results are as follows:1. Abundant secondary metabolism and cell wall degrading enzyme related gene families are lost in P. digitatumThrough the whole genome sequencing, a26Mb high quality P. digitatum genome was obtained. This genome contains103unordered scaffolds, which encodes9,000proteins and162tRNAs. Repeat sequences take1%of the genome. Comparative analysis of PdOl and other Penicillium, Aspergillus and close related phytopathogens indicated that PdOl genome is relatively compact, and encodes smaller number of genes. Synteny and protein family evolution analysis showed that more than100protein families were contracted in PdOl, whereas only2were expanded. Further analysis on gene clusters for secondary metabolite biosynthesis discovered that PdOl lost either key enzymes or the whole clusters for most Penicillium toxins, the only one reserved gene cluster is tryptoquialanine biosynthesis gene cluster. These results explained in molecular perspective that why P. digitatum is unable to produce most toxins. Moreover, an extensive search of carbohydrate active enzymes in PdOl suggested the substantial losses of cell wall degrading enzymes compared with related species, this may in part explain why P. digitatum has a relatively narrow host range.2. Comparative analysis of mitochondrial genome indicated the intron losses during evolutionThe whole mitochondrial genome of PdOl was assembled, which is28,978bp in size with average AT content of74.7%. PdOl mtDNA encodes15proteins and27tRNAs, as well as two ribosome subunits. P. digitatum has similar coding proteins with other species, for example, yeast, but its coding efficiency is much higher (as estimated to80%). The comparison between Penicillium and Aspergillus showed that sequences and orders for most mitochondrial genes are conserved across species, the gene atp8, however, is the most conserved one, which implicated the action of negative selection. Moreover, the introns in mitochondrial genome varies across species, and P. digitatum may have lost the particular introns during evolution.3. Comparative transcriptomics revealed gene expression regulation in carbon source utilization and PacC regulationQuantifying the gene expression changes in Citrus medium and glucose medium defined413genes with290upregulation and123downregualtion. GO annotation of these differential expressed genes showed a broad involvement in secondary metabolism. Many genes participated in carbon source utilization were upregulated, including xylulose reductase, invertase, chitinase, and β-Nacetyl hexosaminidase. In contrast, genes in amino acid or nucleotide metabolism were mostly stable. Furthermore, the whole geneset in tryptoquialanine biosynthesis was upregulated when grown in Citrus medium.Both P. digitatum and Colletotrichum gloeosporioides are able to infect Citrus fruits, but the strategies are opposite. During the infection, P. digitatum produces organic acid to acidify environment, while C. gloeosporioides secretes ammonia to alkalize the infection site. Transcriptome analysis of PacC mutants in both species showed a distincitve regulatory patterns. The PacC binding motif is still conserved in both species, but the regulation direction and magnitude of the targets are distintive. In C. gloeosporioides the number of genes upregulated or downregulated were comparable, but the alkaline dependent genes had higher expression baseline when PacC was activated. However, in P. digitatum, a larger number of genes were repressed in alkaline condition, suggesting that these genes are likely to be activated during infection.4. The newly evolved DMIs resistance is caused by overexpression of PdCYP51B in P. digitatumDuring2000to2010, we collected403P. digitatum strains from major Citrus growing areas in Zhejiang province. Through the sensitivity test to a batch of DMI fungicides, a number of DMIs resistant strains was isolated,89%of them don’t have detectable genotypes. To explore the resistance mechanisms of these strains, the two homologous genes of CYP51were cloned, which were named as PdCYP51B and PdCYP51C. Further analysis on these two genes revealed a unique insertion of199bp fragment in the promoter region of PdCYP51B in IMZ-R3strains. This insert was found neither in IMZ-S nor in IMZV-R1/IMZ-R2strains. Ectopic expression of wild type PdCYP51B decrease sensitivity to DMIs, however, when overexpression of199bp contained PdCYP51B gene in P. digitatum, the DMI resistance was dramatically increased. These data implicated that overexpression of PdCYP51B in IMZ-R3strains is responsible for their DMI resistance.5.199bp insert is a transposable and promoter-like MITEBased on bioinformatic analysis of this199bp insert, a TSD (target site duplication) and a TIR (terminal invert repeat) sites were found to be included in the sequence. This fragment also contains other characteristics, for example, AT rich, unable to code, and can form stable secondary structure. Therefore, we classified this199bp sequence into the miniature inverted-repeat transposable element (MITE) family, and named it as MITE-like element1(PdMLEl). BLAST searching PdMLEl against NCBI database showed no hit matched. Southern blot using PdMLEl as probe in P. digitatum strains and P. expansum as outgroup showed that PdMLEl was abundant in P. digitatum strains but was absent in P. expansum, affiming that PdMLEl is unique to P. digiatatum. The promoter activity of PdMLEl was further confirmed by fusing PdMLEl to the5’-terminus of GFP, the fluorescence was even stronger than trpC fused GFP. Finally, the core element of PdMLEl was limited into20bp region using promoter mapping approach. Getting together, we proposed a regulatory model for transposon induced fungicide resistance in P. digitatum.The studies above are tightly connected, the information provided both for fungicide resistance and genome sequences will shed light on the further functional researches in P. digitatum.