Alternariol as virulence and colonization factor of Alternaria alternata during plant infection.

The filamentous fungus Alternaria alternata is a potent producer of many toxic secondary metabolites, which contaminate food and feed. The most prominent one is the polyketide-derived alternariol (AOH) and its derivative alternariol monomethyl ether (AME). Here, we identified the gene cluster for the biosynthesis of AOH and AME by CRISPR/Cas9-mediated gene inactivation of several biosynthesis genes in A. alternata and heterologous expression of the gene cluster in Aspergillus oryzae. The 15 kb-spanning gene cluster consists of a polyketide synthase gene, pksI, an O-methyltransferase, omtI, a FAD-dependent monooxygenase, moxI, a short chain dehydrogenase, sdrI, a putative extradiol dioxygenase, doxI and a transcription factor gene, aohR. Heterologous expression of PksI in A. oryzae was sufficient for AOH biosynthesis. Co-expression of PksI with different tailoring enzymes resulted in AME, 4-hydroxy-alternariol monomethyl ether (4-OH-AME), altenusin (ALN) and altenuene (ALT). Hence, the AOH cluster is responsible for the production of at least five different compounds. Deletion of the transcription factor gene aohR led to reduced expression of pksI and delayed AOH production, while overexpression led to increased expression of pksI and production of AOH. The pksI-deletion strain displayed reduced virulence on tomato, citrus and apple suggesting AOH and the derivatives as virulence and colonization factors.

Establishment of CRISPR/Cas9 in Alternaria alternata.

The filamentous fungus Alternaria alternata is a potent producer of many secondary metabolites, some of which like alternariol or alternariol-methyl ether are toxic and/or cancerogenic. Many Alternaria species do not only cause post-harvest losses of food and feed, but are aggressive plant pathogens. Despite the great economic importance and the large number of research groups working with the fungus, the molecular toolbox is rather underdeveloped. Gene deletions often result in heterokaryotic strains and therefore, gene-function analyses are rather tedious. In addition, A. alternata lacks a sexual cycle and classical genetic approaches cannot be combined with molecular biological methods. Here, we show that CRISPR/Cas9 can be efficiently used for gene inactivation. Two genes of the melanin biosynthesis pathway, pksA and brm2, were chosen as targets. Several white mutants were obtained after several rounds of strain purification through protoplast regeneration or spore inoculation. Mutation of the genes was due to deletions from 1bp to 1.5kbp. The CRISPR/Cas9 system was also used to inactivate the orotidine-5-phosphate decarboxylase gene pyrG to create a uracil-auxotrophic strain. The strain was counter-selected with fluor-orotic acid and could be re-transformed with pyrG from Aspergillus fumigatus and pyr-4 from Neurospora crassa. In order to test the functioning of GFP, the fluorescent protein was fused to a nuclear localization signal derived from the StuA transcription factor of Aspergillus nidulans. After transformation bright nuclei were visible.

Alternaria alternata transcription factor CmrA controls melanization and spore development.

Melanin is a black pigment widely distributed across the kingdoms, from bacterial to human. The filamentous fungus Alternaria alternata is a typical 'black fungus', which produces melanin in its hyphal and especially its asexual spore cell walls. Its biosynthesis follows the dihydroxynaphthalene (DHN) pathway with 1,8-DHN as an intermediate. Two genes, encoding a polyketide synthase (pksA) and a 1,3,8-trihydroxynaphthalene (THN) reductase (brm2), along with a putative transcription factor, CmrA, comprise a small gene cluster. Here we show that CmrA controls the expression of pksA and brm2, but that it also controls the expression of a scytalone dehydratase encoding gene (brm1) located elsewhere in the genome. The regulatory function of CmrA was shown in a reporter assay system. Al. alternata CmrA was expressed in the filamentous fungus Aspergillus nidulans where it was able to induce the expression of a reporter construct under the control of the putative pksA promoter. This suggests direct binding of CmrA to the promoter of pksA in the heterologous system. Likewise, silencing of cmrA in Al. alternata led to white colonies due to the lack of melanin. In addition, hyphal diameter and spore morphology were changed in the mutant and the number of spores reduced. Silencing of brm2 and inhibition of melanin biosynthesis by tricyclazole largely phenocopied the effects of cmrA silencing, suggesting a novel regulatory function of melanin in morphogenetic pathways.