Complex regulation of secondary metabolism controlling pathogenicity in the phytopathogenic fungus Alternaria alternata.

The filamentous fungus Alternaria alternata includes seven pathogenic variants (pathotypes), which produce different host-selective toxins and cause disease on different plants. The Japanese pear, strawberry and tangerine pathotypes produce AK-toxin, AF-toxin and ACT-toxin, respectively, which have a common structural moiety, 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid (EDA). Here, we identified a new gene, AKT7 (AK-toxin biosynthetic gene 7), from the Japanese pear pathotype, which encodes a cytochrome P450 monooxygenase and functions to limit AK-toxin production. AKT7 homologs were found in the strawberry pathotype, but not the tangerine pathotype. However, the strawberry pathotype homolog appeared to include a premature stop codon. Although the Japanese pear pathotype strain has multiple copies of AKT7, a single-copy disruption resulted in mutants with increased production of AK-toxin and EDA. AKT7 overexpression in the three pathotypes caused marked reductions of toxin and EDA production, suggesting that Akt7 catalyzes a side reaction of EDA or its precursor. AKT7 overexpression caused reduced virulence in these pathotypes. We also found that AKT7 transcripts predominantly include misspliced mRNAs, which have premature stop codons. Our observations suggest that the AK-toxin production required for full virulence is regulated in a complex way by the copy number and intron information content of AKT7.

GIGAS CELL1, a novel negative regulator of the anaphase-promoting complex/cyclosome, is required for proper mitotic progression and cell fate determination in Arabidopsis.

Increased cellular ploidy is widespread during developmental processes of multicellular organisms, especially in plants. Elevated ploidy levels are typically achieved either by endoreplication or endomitosis, which are often regarded as modified cell cycles that lack an M phase either entirely or partially. We identified GIGAS CELL1 (GIG1)/OMISSION OF SECOND DIVISION1 (OSD1) and established that mutation of this gene triggered ectopic endomitosis. On the other hand, it has been reported that a paralog of GIG1/OSD1, UV-INSENSITIVE4 (UVI4), negatively regulates endoreplication onset in Arabidopsis thaliana. We showed that GIG1/OSD1 and UVI4 encode novel plant-specific inhibitors of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. These proteins physically interact with APC/C activators, CDC20/FZY and CDH1/FZR, in yeast two-hybrid assays. Overexpression of CDC20.1 and CCS52B/FZR3 differentially promoted ectopic endomitosis in gig1/osd1 and premature occurrence of endoreplication in uvi4. Our data suggest that GIG1/OSD1 and UVI4 may prevent an unscheduled increase in cellular ploidy by preferentially inhibiting APC/C(CDC20) and APC/C(FZR), respectively. Generation of cells with a mixed identity in gig1/osd1 further suggested that the APC/C may have an unexpected role for cell fate determination in addition to its role for proper mitotic progression.

Roles of GIG1 and UVI4 in genome duplication in Arabidopsis thaliana.

Endomitosis and endoreplication are atypical modes of cell cycle that results in genome duplication in single nucleus. Because the cell size of given cell type is generally proportional to the nuclear DNA content, endoreplication and endomitosis are effective strategy of cell growth, which are widespread in multicellular organisms, especially those in plant kingdom. We found that these processes might be differently regulated by GIGAS CELL1 (GIG1) and its paralog UV-INSENSITIVE4 (UVI4) in Arabidopsis thaliana. GIG1 and UVI4 may negatively regulate activities of anaphase-promoting complex or cyclosome (APC/C) ubiquitin ligase that acts as an important mitotic regulator. The gig1 mutation induced ectopic occurrence of endomitosis during somatic cell division, while it has been reported that uvi4 mutation resulted in premature occurrence of endoreplication during organ development. Overexpression of GIG1 and UVI4 dramatically increased the amount of mitotic cyclin, CYCB1;1, a well-known substrate of APC/C. Ectopic endomitosis in gig1 was enhanced by mutation in CYCB2;2 and suppressed by downregulation of APC10 encoding a core subunit of APC/C. Overexpression of CDC20.1, an activator protein of APC/C, further promoted the ectopic endomitosis in gig1. These findings suggest that endomitosis and endoreplication are regulated by similar molecular mechanisms, in which two related proteins, GIG1 and UVI4, may inhibit APC/C in different ways.