Fungal NRPS-PKS Hybrid Enzymes Biosynthesize New γ-Lactam Compounds, Taslactams A-D, Analogous to Actinomycete Proteasome Inhibitors.

Proteasome inhibitors with γ-lactam structure, such as lactacystin and salinosporamide A, have been isolated from actinomycetes and have attracted attention as lead compounds for anticancer drugs. Previously, we identified a unique enzyme TAS1, which is the first reported fungal NRPS-PKS hybrid enzyme, from the filamentous fungus Pyricularia oryzae for the biosynthesis of a mycotoxin tenuazonic acid, a tetramic acid compound without γ-lactam structure. Homologues of TAS1 have been identified in several fungal genomes and classified into four groups (A-D). Here, we show that the group D TAS1 homologues from two filamentous fungi can biosynthesize γ-lactam compounds, taslactams A-D, with high similarity to actinomycete proteasome inhibitors. One of the γ-lactam compounds, taslactam C, showed potent proteasome inhibitory activity. In contrast to actinomycete γ-lactam compounds which require multiple enzymes for biosynthesis, the TAS1 homologue alone was sufficient for the biosynthesis of the fungal γ-lactam compounds.

CmLec4, a lectin from the fungus Cordyceps militaris, controls host infection and fruiting body formation.

Fungi belonging to the Ascomycete genus Cordyceps are endoparasitoids and parasites, mainly of insects and other arthropods. Cordyceps militaris has been used as a therapeutic drug for cancer patients. However, the infection, parasitism, and fruiting body formation mechanisms of this fungus are still unknown. Based on our hypothesis that lectin(s) is involved in the interaction between the C. militaris fungi and insects, we partially purified and characterized a new lectin from C. militaris, designated CmLec4. In addition, we searched for substance(s) in the infected silkworm extracts that could bind to CmLec4, and succeeded in purifying the sex-specific storage protein 2 as a specific binding target. To examine function of the binding protein during the process of parasitism, we investigated the effect of recombinant CmLec4 on silkworms by inoculating the protein into silkworm pupae, and found that it significantly delayed emergence compared to the control. Furthermore, cmlec4 gene knockout strains constructed in this study produced markedly lower amounts of fruiting body than the wild-type strain. All the results revealed that the lectin CmLec4 produced by C. militaris would be involved in the infection into silkworm and fruiting body formation from the host.

Tenuazonic acid production is dispensable for virulence, but its biosynthetic gene expression pattern is associated with the infection of Pyricularia oryzae.

Tenuazonic acid (TeA) is a toxin produced by the rice blast fungus Pyricularia oryzae. Although knockout of the TeA biosynthetic gene TAS1 did not affect the virulence of P. oryzae, constitutive TAS1 expression suppressed its infection. TAS1 expression was induced alongside transition of P. oryzae infection behavior. The results suggested that controlling TeA biosynthesis is important for P. oryzae infection.

High-quality genome assembly of the soybean fungal pathogen Cercospora kikuchii.

Plant diseases caused by the Cercospora genus of ascomycete fungi are a major concern for commercial agricultural practices. Several Cercospora species can affect soybeans, such as Cercospora kikuchii which causes soybean leaf blight. Speciation in Cercospora on soybean has not been adequately studied. Some cryptic groups of Cercospora also cause diseases on soybean. Moreover, it has been known C. kikuchii population genetic structure is different between countries. Consequently, further genomic information could help to elucidate the covert differentiation of Cercospora diseases in soybean. Here, we report for the first time, a chromosome-level genome assembly for C. kikuchii. The genome assembly of 9 contigs was 34.44 Mb and the N50 was 4.19 Mb. Based on ab initio gene prediction, several candidates for pathogenicity-related genes, including 242 genes for putative effectors, 55 secondary metabolite gene clusters, and 399 carbohydrate-active enzyme genes were identified. The genome sequence and the features described in this study provide a solid foundation for comparative and evolutionary genomic analysis for Cercospora species that cause soybean diseases worldwide.

Near-isogenic soybean lines carrying Asian soybean rust resistance genes for practical pathogenicity validation.

Asian soybean rust caused by the fungal pathogen Phakopsora pachyrhizi is the most devastating disease of soybean. The host cultivar specificity of the pathogen shows considerable differentiation depending on the area and season of its emergence. Although resistance genes for P. pachyrhizi (Rpp) have been reported in several soybean varieties, the genetic background of these varieties is highly differentiated. Furthermore, some of the varieties harbor unknown genetic factors in addition to Rpp that could influence resistance reactions against the pathogen. In order to gain a comprehensive understanding of Rpp-P. pachyrhizi interactions, homogenous plant material harboring Rpp genes is necessary. In this study, we bred Rpp-near isogenic lines (Rpp-NILs), which retained identical plant characters originating from a single genetic background, and accordingly showed low-variant compatible/incompatible reactions against the pathogen. These Rpp-NILs can be used as genetic resources for studying P. pachyrhizi epidemiology and elucidating resistance mechanisms. Compatible/incompatible relationships between the soybean rust resistance gene Rpp and isolates of the pathogen P. pachyrhizi are clearly distinguishable using the Rpp-NILs bred in this study.

Sequencing of individual chromosomes of plant pathogenic Fusarium oxysporum.

A small chromosome in reference isolate 4287 of F. oxysporum f. sp. lycopersici (Fol) has been designated as a 'pathogenicity chromosome' because it carries several pathogenicity related genes such as the Secreted In Xylem (SIX) genes. Sequence assembly of small chromosomes in other isolates, based on a reference genome template, is difficult because of karyotype variation among isolates and a high number of sequences associated with transposable elements. These factors often result in misassembly of sequences, making it unclear whether other isolates possess the same pathogenicity chromosome harboring SIX genes as in the reference isolate. To overcome this difficulty, single chromosome sequencing after Contour-clamped Homogeneous Electric Field (CHEF) separation of chromosomes was performed, followed by de novo assembly of sequences. The assembled sequences of individual chromosomes were consistent with results of probing gels of CHEF separated chromosomes with SIX genes. Individual chromosome sequencing revealed that several SIX genes are located on a single small chromosome in two pathogenic forms of F. oxysporum, beyond the reference isolate 4287, and in the cabbage yellows fungus F. oxysporum f. sp. conglutinans. The particular combination of SIX genes on each small chromosome varied. Moreover, not all SIX genes were found on small chromosomes; depending on the isolate, some were on big chromosomes. This suggests that recombination of chromosomes and/or translocation of SIX genes may occur frequently. Our method improves sequence comparison of small chromosomes among isolates.

A new biotype of Fusarium oxysporum f. sp. lycopersici race 2 emerged by a transposon-driven mutation of avirulence gene AVR1.

Emergence of races in Fusarium oxysporum f. sp. lycopersici (Fol) is caused by loss or mutation of at least one avirulence (AVR) gene. The product of AVR1 is a small protein (Avr1) secreted by Fol in tomato xylem sap during infection. This protein triggers Fol race 1 specific resistance (I) in tomato, indicating that AVR1 is an AVR gene. Deletion of AVR1 in race 1 resulted in the emergence of race 2, and an additional mutation in AVR2 generated race 3. Previously, we reported a new biotype of race 3, KoChi-1, in which AVR1 was truncated by a transposon Hormin, which suggested a new route to evolution of races in Fol However, to date no race 2 isolate carrying Hormin-truncated AVR1 has been reported. In this report, we describe such isolates, represented by Chiba-5, in which Hormin insertion occurred in AVR1 at a position different from that in KoChi-1. AVR1 truncation in both isolates resulted in production of defective Avr1 proteins. Chiba-5 and KoChi-1 belong to different phylogenetic clades, A1 and A2, respectively, suggesting that insertion of Hormin in AVR1 in Chiba-5 and KoChi-1 occurred as independent evolutionary events.

The tomato wilt fungus Fusarium oxysporum f. sp. lycopersici shares common ancestors with nonpathogenic F. oxysporum isolated from wild tomatoes in the Peruvian Andes.

Fusarium oxysporum is an ascomycetous fungus that is well-known as a soilborne plant pathogen. In addition, a large population of nonpathogenic F. oxysporum (NPF) inhabits various environmental niches, including the phytosphere. To obtain an insight into the origin of plant pathogenic F. oxysporum, we focused on the tomato (Solanum lycopersicum) and its pathogenic F. oxysporum f. sp. lycopersici (FOL). We collected F. oxysporum from wild and transition Solanum spp. and modern cultivars of tomato in Chile, Ecuador, Peru, Mexico, Afghanistan, Italy, and Japan, evaluated the fungal isolates for pathogenicity, VCG, mating type, and distribution of SIX genes related to the pathogenicity of FOL, and constructed phylogenies based on ribosomal DNA intergenic spacer sequences. All F. oxysporum isolates sampled were genetically more diverse than FOL. They were not pathogenic to the tomato and did not carry SIX genes. Certain NPF isolates including those from wild Solanum spp. in Peru were grouped in FOL clades, whereas most of the NPF isolates were not. Our results suggested that the population of NPF isolates in FOL clades gave rise to FOL by gaining pathogenicity.