Lutein, a Natural Carotenoid, Induces α-1,3-Glucan Accumulation on the Cell Wall Surface of Fungal Plant Pathogens.

α-1,3-Glucan, a component of the fungal cell wall, is a refractory polysaccharide for most plants. Previously, we showed that various fungal plant pathogens masked their cell wall surfaces with α-1,3-glucan to evade plant immunity. This surface accumulation of α-1,3-glucan was infection specific, suggesting that plant factors might induce its production in fungi. Through immunofluorescence observations of fungal cell walls, we found that carrot (Daucus carota) extract induced the accumulation of α-1,3-glucan on germlings in Colletotrichum fioriniae, a polyphagous fungal pathogen that causes anthracnose disease in various dicot plants. Bioassay-guided fractionation of carrot leaf extract successfully identified two active substances that caused α-1,3-glucan accumulation in this fungus: lutein, a carotenoid widely distributed in plants, and stigmasterol, a plant-specific membrane component. Lutein, which had a greater effect on C. fioriniae, also induced α-1,3-glucan accumulation in other Colletotrichum species and in the phylogenetically distant rice pathogen Cochliobolus miyabeanus, but not in the rice pathogen Magnaporthe oryzae belonging to the same phylogenetic subclass as Colletotrichum. Our results suggested that fungal plant pathogens reorganize their cell wall components in response to specific plant-derived compounds, which these pathogens may encounter during infection.

Functional analysis of the α-1,3-glucan synthase genes agsA and agsB in Aspergillus nidulans: agsB is the major α-1,3-glucan synthase in this fungus.

Although α-1,3-glucan is one of the major cell wall polysaccharides in filamentous fungi, the physiological roles of α-1,3-glucan remain unclear. The model fungus Aspergillus nidulans possesses two α-1,3-glucan synthase (AGS) genes, agsA and agsB. For functional analysis of these genes, we constructed several mutant strains in A. nidulans: agsA disruption, agsB disruption, and double-disruption strains. We also constructed several CagsB strains in which agsB expression was controlled by the inducible alcA promoter, with or without the agsA-disrupting mutation. The agsA disruption strains did not show markedly different phenotypes from those of the wild-type strain. The agsB disruption strains formed dispersed hyphal cells under liquid culture conditions, regardless of the agsA genetic background. Dispersed hyphal cells were also observed in liquid culture of the CagsB strains when agsB expression was repressed, whereas these strains grew normally in plate culture even under the agsB-repressed conditions. Fractionation of the cell wall based on the alkali solubility of its components, quantification of sugars, and (13)C-NMR spectroscopic analysis revealed that α-1,3-glucan was the main component of the alkali-soluble fraction in the wild-type and agsA disruption strains, but almost no α-1,3-glucan was found in the alkali-soluble fraction derived from either the agsB disruption strain or the CagsB strain under the agsB-repressed conditions, regardless of the agsA genetic background. Taken together, our data demonstrate that the two AGS genes are dispensable in A. nidulans, but that AgsB is required for normal growth characteristics under liquid culture conditions and is the major AGS in this species.

Surface α-1,3-glucan facilitates fungal stealth infection by interfering with innate immunity in plants.

Plants evoke innate immunity against microbial challenges upon recognition of pathogen-associated molecular patterns (PAMPs), such as fungal cell wall chitin. Nevertheless, pathogens may circumvent the host PAMP-triggered immunity. We previously reported that the ascomycete Magnaporthe oryzae, a famine-causing rice pathogen, masks cell wall surfaces with α-1,3-glucan during invasion. Here, we show that the surface α-1,3-glucan is indispensable for the successful infection of the fungus by interfering with the plant's defense mechanisms. The α-1,3-glucan synthase gene MgAGS1 was not essential for infectious structure development but was required for infection in M. oryzae. Lack or degradation of surface α-1,3-glucan increased fungal susceptibility towards chitinase, suggesting the protective role of α-1,3-glucan against plants' antifungal enzymes during infection. Furthermore, rice plants secreting bacterial α-1,3-glucanase (AGL-rice) showed strong resistance not only to M. oryzae but also to the phylogenetically distant ascomycete Cochlioborus miyabeanus and the polyphagous basidiomycete Rhizoctonia solani; the histocytochemical analysis of the latter two revealed that α-1,3-glucan also concealed cell wall chitin in an infection-specific manner. Treatment with α-1,3-glucanase in vitro caused fragmentation of infectious hyphae in R. solani but not in M. oryzae or C. miyabeanus, indicating that α-1,3-glucan is also involved in maintaining infectious structures in some fungi. Importantly, rapid defense responses were evoked (a few hours after inoculation) in the AGL-rice inoculated with M. oryzae, C. miyabeanus and R. solani as well as in non-transgenic rice inoculated with the ags1 mutant. Taken together, our results suggest that α-1,3-glucan protected the fungal cell wall from degradative enzymes secreted by plants even from the pre-penetration stage and interfered with the release of PAMPs to delay innate immune defense responses. Because α-1,3-glucan is nondegradable in plants, it is reasonable that many fungal plant pathogens utilize α-1,3-glucan in the innate immune evasion mechanism and some in maintaining the structures.

Kojic acid biosynthesis in Aspergillus oryzae is regulated by a Zn(II)(2)Cys(6) transcriptional activator and induced by kojic acid at the transcriptional level.

A gene encoding the Zn(II)(2)Cys(6) transcriptional factor is clustered with two genes involved in biosynthesis of a secondary metabolite, kojic acid (KA), in Aspergillus oryzae. We determined that the gene was essential for KA production and the transcriptional activation of KA biosynthetic genes, which were triggered by the addition of KA.

Penicillin biosynthesis in Aspergillus oryzae and its overproduction by genetic engineering.

Aspergillus oryzae penicillin biosynthetic genes were clustered. The penicillin production was positively regulated by VeA, a global gene regulator required for transcriptional expression of the penicillin biosynthetic genes. Overexpression of the biosynthetic genes by a strong promoter yielded a greater than 100-fold increase in penicillin production.

Mstu1, an APSES transcription factor, is required for appressorium-mediated infection in Magnaporthe grisea.

The APSES protein family includes important transcriptional regulators of morphological processes in ascomycetes. We identified a deletion mutant of the APSES protein Mstu1 in Magnaporthe grisea that showed reduced conidiation and mycelial growth. Mstu1 formed a number of appressoria comparable to the wild type, although appressorium formation was delayed. In M. grisea, rapid transfer of conidial glycogen and lipid droplets to incipient appressoria is required for appressorial turgor generation, which the fungus uses to penetrate plant cuticles. Appressorial turgor was low in mstu1 and the mutant was deficient in appressorium-mediated invasion of rice leaves. The transfer of conidial glycogen and lipid droplets was remarkably delayed in mstu1, and a consequent delay in degradation of these conidial reserves was observed. Our results indicate that Mstu1 is required for appressorium-mediated infection due to its involvement in the mobilization of lipids and glycogen.

Dynamics of cell wall components of Magnaporthe grisea during infectious structure development.

Oligosaccharides derived from cell wall of fungal pathogens induce host primary immune responses. To understand fungal strategies circumventing the host plant immune responses, cell wall polysaccharide localization was investigated using fluorescent labels during infectious structure differentiation in the rice blast fungus Magnaporthe grisea. alpha-1,3-glucan was labelled only on appressoria developing on plastic surfaces, whereas it was detected on both germ tubes and appressoria on plant surfaces. Chitin, chitosan and beta-1,3-glucan were detected on germ tubes and appressoria regardless of the substrate. Major polysaccharides labelled at accessible surface of infectious hyphae were alpha-1,3-glucan and chitosan, but after enzymatic digestion of alpha-1,3-glucan, beta-1,3-glucan and chitin became detectable. Immunoelectron microscopic analysis showed alpha-1,3-glucan and beta-1,3-glucan intermixed in the cell wall of infectious hyphae; however, alpha-1,3-glucan tended to be distributed farther from the fungal cell membrane. The fungal cell wall became more tolerant to chitinase digestion upon accumulation of alpha-1,3-glucan. Accumulation of alpha-1,3-glucan was dependent on the Mps1 MAP kinase pathway, which was activated by a plant wax derivative, 1,16-hexadecanediol. Taken together, alpha-1,3-glucan spatially and functionally masks beta-1,3-glucan and chitin in the cell wall of infectious hyphae. Thus, a dynamic change of composition of cell wall polysaccharides occurs during plant infection in M. grisea.

Differential responses of rice to inoculation with wild-type and non-pathogenic mutants of Magnaporthe oryzae.

We analyzed the response of rice to Magnaporthe oryzae infection using two mutant strains deficient in Mgb1 and Mst12, which are essential for the development of appresoria and penetration pegs. Both mutants induced the much lower levels of accumulation of phytoalexins than wild-type, suggesting that the massive production of phytoalexins requires the fungal invasion of rice cells. Intense accumulation of H2O2 in a single whole cell also required fungal penetration. Microarray analysis of rice gene expression revealed mutant-specific gene expression, indicating that signal exchange between rice and M. oryzae commence before fungal penetration of the rice cell. In situ detection of mRNAs for peroxidase and beta-1,3-glucanase showed that expression of these genes also occurs after penetration as observed for phytoalexin production.

Genome-wide validation of Magnaporthe grisea gene structures based on transcription evidence.

Accurate cDNA data is useful to validate gene structures in a genome. We sequenced 35189 expressed sequence tags (ESTs) obtained from the highly destructive rice blast fungus, Magnaporthe grisea. Our custom-made computational programs mapped these ESTs on the M. grisea genome sequence, and reconstructed gene structures as well as protein-coding regions. As a result, we predicted 4480 protein-coding sequences, which were more accurate than ab initio predictions. Moreover, cross-species comparisons suggested that our predicted proteins were nearly complete. The cDNA clones obtained in this study will be important for further experimental studies. Our genome annotation is available at http://www.mg.dna.affrc.go.jp/.

MgLig4, a homolog of Neurospora crassa Mus-53 (DNA ligase IV), is involved in, but not essential for, non-homologous end-joining events in Magnaporthe grisea.

In many eukaryotic organisms, the non-homologous end-joining (NHEJ) system is a major pathway for the repair of DNA double-strand breaks (DSBs). DNA ligase IV is a component of the NHEJ system and is strictly required for the NHEJ system in Saccharomyces cerevisiae and in Neurospora crassa. To investigate the functions of DNA Ligase IV in Magnaporthe grisea, we generated deletion mutants of MGLIG4, which encodes a homolog of N. crassa DNA Ligase IV. Mutants (mglig4) showed no defects in asexual or sexual growth, and were fully pathogenic. Compared to the wild-type, mglig4 exhibited weak sensitivity to a DNA-damaging agent, camptothecin. In addition, the frequency of targeted-gene replacement was relatively elevated in mglig4, although this varied in a gene-dependent manner. Surprisingly, non-homologous integration of DNA was frequently observed in mglig4 transformants. Our results demonstrate that MgLig4 is involved in, but not essential for, the NHEJ system in M. grisea.

Construction of a binary vector for knockout and expression analysis of rice blast fungus genes.

We developed an efficient method to analyze gene function and expression of the rice blast fungus. We constructed a GATEWAY binary vector, which generates a gene-targeted disruptant carrying a green fluorescent protein gene under the native promoter of the target gene. Using this method, the knockout efficiency and expression patterns of two hypothetical genes were determined.

Two PAK kinase genes, CHM1 and MST20, have distinct functions in Magnaporthe grisea.

In the rice blast fungus Magnaporthe grisea, the Pmk1 mitogen-activated protein (MAP) kinase is essential for appressorium formation and infectious growth. PMK1 is homologous to yeast Fus3 and Kss1 MAP kinases that are known to be regulated by the Ste20 PAK kinase for activating the pheromone response and filamentation pathways. In this study, we isolated and characterized two PAK genes, CHM1 and MST20, in M. grisea. Mutants disrupted in MST20 were reduced in aerial hyphae growth and conidiation, but normal in growth rate, appressorium formation, penetration, and plant infection. In chm1 deletion mutants, growth, conidiation, and appressorium formation were reduced significantly. Even though appressoria formed by chm1 mutants were defective in penetration, chm1 mutants were able to grow invasively on rice leaves and colonize through wounds. The chm1 mutants were altered in conidiogenesis and produced conidia with abnormal morphology. Hyphae of chm1 mutants had normal septation, but the length of hyphal compartments was reduced. On nutritionally poor oatmeal agar, chm1 mutants were unstable and produced sectors that differed from original chm1 mutants in growth rate, conidiation, or colony morphology. However, none of the monoconidial cultures derived from these spontaneous sectors were normal in appressorial penetration and fungal pathogenesis. These data suggest that MST20 is dispensable for plant infection in M. grisea, but CHM1 plays a critical role in appressorium formation and penetration. Both mst20 and chm1 deletion mutants were phenotypically different from the pmk1 mutant that is defective in appressorium formation and infectious hyphae growth. It is likely that MST20 and CHM1 individually play no critical role in activating the PMK1 MAP kinase pathway during appressorium formation and infectious hyphae growth. However, CHM1 appears to be essential for appressorial penetration and CHM1 and MST20 may have redundant functions in M. grisea.

The G-beta subunit MGB1 is involved in regulating multiple steps of infection-related morphogenesis in Magnaporthe grisea.

Trimeric G-proteins transmit extracellular signals to various downstream effectors (e.g. MAP kinases) in eukaryotes. In the rice blast fungus Magnaporthe grisea, the Pmk1 MAP kinase is essential for appressorium formation and infectious growth. The pmk1 deletion mutant fails to form appressoria but still responds to exogenous cAMP for tip deformation. Since gene disruption mutants of three Galpha subunits still form appressoria and are phenotypically different from pmk1 mutants, it is likely that the Pmk1 pathway is activated by Gbeta in M. grisea. In this study, we isolated and characterized the MGB1 gene that encodes the G subunit in M. grisea. Mutants disrupted in MGB1 were reduced in conidiation. Conidia from mgb1 mutants were defective in appressorium formation and failed to penetrate or grow invasively on rice leaves. Exogenous cAMP induced appressorium formation in mgb1 mutants, but these appressoria were abnormal in shape and could not penetrate. The intracellular cAMP level was reduced in mgb1 mutants and the defects in conidiation and hyphal growth were partially suppressed with 1 mM cAMP. Transformants expressing multiple copies of MGB1 were able to form appressoria on hydrophilic surfaces. Our results suggest that MGB1 may be involved in the cAMP signalling for regulating conidiation, surface recognition and appressorium formation. The Pmk1 pathway may be the downstream target of MGB1 for regulating penetration and infectious hyphae growth in M. grisea.