Endocytic protein Pal1 regulates appressorium formation and is required for full virulence of Magnaporthe oryzae

Endocytosis plays key roles during infection of plant-pathogenic fungi, but its regulatory mechanisms are still largely unknown. Here, we identified a putative endocytosis-related gene, PAL1, which was highly expressed in appressorium of Magnaporthe oryzae, and was found to be important for appressorium formation and maturation. Deletion of PAL1 significantly reduced the virulence of M. oryzae due to defects in appressorial penetration and invasive growth in host cells. The Pal1 protein interacted and colocalized with the endocytosis protein Sla1, suggesting it is involved in endocytosis. The Δpal1 mutant was significantly reduced in appressorium formation, which was recovered by adding exogenous cAMP and 3-isobutyl-1-methylxanthine (IBMX). Moreover, the phosphorylation level of Pmk1 in Δpal1 was also reduced, suggesting Pal1 functions upstream of both the cAMP and Pmk1 signalling pathways. As a consequence, the utilization of glycogen and lipid, appressorial autophagy, actin ring formation, localization of septin proteins, as well as turgor accumulation were all affected in the Δpal1 mutant. Taken together, Pal1 regulates cAMP and the Pmk1 signalling pathway for appressorium formation and maturation to facilitate infection of M. oryzae.     

A novel gene MGA1 is required for appressorium formation in Magnaporthe grisea

Insertional mutagenesis is an effective way to study the infection mechanism of fungal pathogens. In an attempt to identify the genes involved in appressorium formation from Magnaporthe grisea, we carried out Agrobacterium tumefaciens mediated transformation (ATMT) of the fungus. Analysis of the region flanking the T-DNA integration site in one of the appressorium mutants showed insertion in a gene coding a 78 amino acid protein (MGA1), showing no significant homology to any of the known proteins. The mutant mga1 caused neither foliar nor root infection. Complementation of the mutated gene with the full length wild type gene restored appressorium formation as well as rice infection demonstrating the involvement of this gene in pathogenicity of M. grisea. In an indirect immunolocalisation assay, the MGA1 expression was seen predominantly in germ tube and appressoria. The mutant was impaired in glycogen and lipid mobilization required for appressorium formation. The glycerol content in the mycelia of the mutant under hyperosmotic stress conditions was less as compared to wild type and was thus unable to tolerate the hyperosmotic stress induced by sorbitol. We hypothesize that MGA1 plays a crucial role in signal transduction leading to the metabolism of glycogen and lipids, which is a part of appressorium differentiation process.     

The Magnaporthe grisea snodprot1 homolog, MSP1, is required for virulence

Secreted proteins play central roles in plant-microbe interactions acting as signals, toxins, and effectors. One important group of small secreted proteins is the snodprot1 family, members of which have demonstrated phytotoxic properties. A split-marker transformation system was applied for gene deletion of the snodprot1 homolog, MSP1, in the rice blast fungus Magnaporthe grisea. msp1 mutants were phenotypically indistinguishable from wild type and elaborated apparently normal appressoria. However, the deletion mutants were greatly reduced in virulence primarily due to impaired growth in planta. Western blot analysis showed that the protein was secreted and not associated with the fungal cell wall. When purified MSP1 protein was applied to wounded leaf tissue, no apparent phytotoxic effects were noted. This is the first report to the authors' knowledge that directly implicates a snodprot1 protein as a virulence factor.     

Magnaporthe grisea pth11p is a novel plasma membrane protein that mediates appressorium differentiation in response to inductive substrate cues.

Mutagenesis of Magnaporthe grisea strain 4091-5-8 led to the identification of PTH11, a pathogenicity gene predicted to encode a novel transmembrane protein. We localized a Pth11-green fluorescent protein fusion to the cell membrane and vacuoles. pth11 mutants of strain 4091-5-8 are nonpathogenic due to a defect in appressorium differentiation. This defect is reminiscent of wild-type strains on poorly inductive surfaces; conidia germinate and undergo early differentiation events, but appressorium maturation is impaired. Functional appressoria are formed by pth11 mutants at 10 to 15% of wild-type frequencies, suggesting that the protein encoded by PTH11 (Pth11p) is not required for appressorium morphogenesis but is involved in host surface recognition. We assayed Pth11p function in multiple M. grisea strains. These experiments indicated that Pth11p can activate appressorium differentiation in response to inductive surface cues and repress differentiation on poorly inductive surfaces and that multiple signaling pathways mediate differentiation. PTH11 genes from diverged M. grisea strains complemented the 4091-5-8 pth11 mutant, indicating functional conservation. Exogenous activation of cellular signaling suppressed pth11 defects. These findings suggest that Pth11p functions at the cell cortex as an upstream effector of appressorium differentiation in response to surface cues.     

A Rho3 homolog is essential for appressorium development and pathogenicity of Magnaporthe grisea

The small GTPase Rho3 is conserved in fungi and plays a key role in the control of cell polarity and exocytosis in yeast. In this report, we show that a Rho3 homolog, MgRho3, is dispensable for polarized hyphal growth in the rice blast fungus Magnaporthe grisea. However, MgRho3 is required for plant infection. Appressoria formed by the Mgrho3 deletion mutants are morphologically abnormal and defective in plant penetration. Conidia of the Mgrho3 deletion mutants are narrower than those of the wild-type strain and delayed in germination. Transformants expressing a dominant negative Mgrho3 allele exhibit similar phenotypes as the Mgrho3 deletion mutant, while transformants expressing a constitutively active allele of MgRho3 can produce normal conidia but remain defective in appressorium formation and plant infection. In contrast, overexpression of wild-type MgRho3 increases the infectivity of M. grisea. Our results reveal a new role for the conserved Rho3 as a critical regulator of developmental processes and pathogenicity of M. grisea.     

Peroxisomal fission is induced during appressorium formation and is required for full virulence of the rice blast fungus

Peroxisomes are involved in various metabolic processes and are important for virulence in different pathogenic fungi. How peroxisomes rapidly emerge in the appressorium during fungal infection is poorly understood. Here, we describe a gene, PEF1, which can regulate peroxisome formation in the appressorium by controlling peroxisomal fission, and is required for plant infection in the rice blast fungus Magnaporthe oryzae. Targeted deletion of PEF1 resulted in a reduction in virulence and a delay in penetration and invasive growth in host cells. PEF1 was particularly expressed during appressorial development, and its encoding protein was co‐localized with peroxisomes during appressorial development. Compared with the massive vesicle‐shaped peroxisomes formed in the wild‐type appressorium, the Δpef1 mutant could only form stringy linked immature peroxisomes, suggesting that PEF1 was involved in peroxisomal fission during appressorium formation. We also found that the Δpef1 mutant could not utilize fatty acids efficiently, which can improve significantly the expression level of PEF1 and induce peroxisomal fission. As expected, the Δpef1 mutant showed reduced intracellular production of reactive oxygen species (ROS) during appressorium formation and induced ROS accumulation in host cells during infection. Taken together, PEF1‐mediated peroxisomal fission is important for fungal infection by controlling the number of peroxisomes in the appressorium.     

A fungal metallothionein is required for pathogenicity of Magnaporthe grisea

The causal agent of rice blast disease, the ascomycete fungus Magnaporthe grisea, infects rice (Oryza sativa) plants by means of specialized infection structures called appressoria, which are formed on the leaf surface and mechanically rupture the cuticle. We have identified a gene, Magnaporthe metallothionein 1 (MMT1), which is highly expressed throughout growth and development by M. grisea and encodes an unusual 22-amino acid metallothionein-like protein containing only six Cys residues. The MMT1-encoded protein shows a very high affinity for zinc and can act as a powerful antioxidant. Targeted gene disruption of MMT1 produced mutants that show accelerated hyphal growth rates and poor sporulation but had no effect on metal tolerance. Mmt1 mutants are incapable of causing plant disease because of an inability to bring about appressorium-mediated cuticle penetration. Mmt1 appears to be distributed in the inner side of the cell wall of the fungus. These findings indicate that Mmt1-like metallothioneins may play a novel role in fungal cell wall biochemistry that is required for fungal virulence.     

The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea.

Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection cell, an appressorium, that is required for infection of its host. Previously, cAMP was implicated in the endogenous signaling pathway leading to appressorium formation. To obtain direct evidence for the role of cAMP in appressorium formation, the gene encoding the catalytic subunit of the cAMP-dependent protein kinase (cpkA) was cloned, sequenced, and disrupted. Polymerase chain reaction primers designed after highly conserved regions in the same gene from other organisms were used to amplify genomic DNA fragments. The cloned amplification products were used to identify genomic clones. DNA blot analysis indicated that cpkA is present as a single copy in the genome. cpkA consists of 1894 bp, including three short introns sufficient to encode a protein of 539 amino acids with a predicted molecular mass of 60.7 kD. The deduced peptide shares > 45% identity with other catalytic subunits and contains all functional motifs and residues with the addition of a glutamine-rich region at the N terminus. Two transformants, L5 and T-182, in which cpkA had been replaced with a hygromycin resistance gene cassette, were unable to produce appressoria, could not be induced to form appressoria by cAMP, and were nonpathogenic on susceptible rice, even when leaves were abraded. These results were confirmed by analysis of 57 progeny from a cross between transformant L5 and the wild-type laboratory strain 70-6. Other aspects of growth and development, including vegetative growth as well as asexual and sexual competence, were unaffected when measured in vitro. These results provide direct evidence that the cAMP-dependent protein kinase is necessary for infection-related morphogenesis and pathogenesis in a phytopathogenic fungus.     

Peroxisomal carnitine acetyl transferase is required for elaboration of penetration hyphae during plant infection by Magnaporthe grisea

Magnaporthe grisea, the causal agent of rice blast disease, invades plant tissue due to the action of specialized infection structures called appressoria, which are used to breach the leaf cuticle and allow development of intracellular, infectious hyphae. In this report we demonstrate that peroxisomal carnitine acetyl transferase (CAT) activity is necessary for appressorium function, and in particular, for the elaboration of primary penetration hyphae. The major CAT activity in M. grisea is encoded by the PTH2 gene, which shows elevated expression in response to acetate and lipid, and is regulated by the cyclic AMP response pathway. Furthermore, a Pth2-GFP fusion protein colocalizes with a peroxisomal marker protein. Targeted deletion of PTH2, generated mutants that were completely non-pathogenic, lacked CAT activity and were unable to utilize a range of lipid substrates. The impairment of appressorium function in Deltapth2 was associated with a delay in lipid reserve mobilization from germ tubes into developing infection cells, and abnormal chitin distribution in infection structures. Addition of glucose to Deltapth2 mutants partially restored the ability to cause rice blast disease and lipid reserve mobilization. Taken together, our findings provide evidence that Pth2 plays a role in the generation of acetyl CoA pools necessary for appressorium function and rapid elaboration of penetration hyphae during host infection.     

The glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic fungus Magnaporthe grisea

We describe the isolation and characterization of ICL1 from the rice blast fungus Magnaporthe grisea, a gene that encodes isocitrate lyase, one of the principal enzymes of the glyoxylate cycle. ICL1 shows elevated expression during development of infection structures and cuticle penetration, and a targeted gene replacement showed that the gene is required for full virulence by M. grisea. In particular, we found that the prepenetration stage of development, before entry into plant tissue, is affected by loss of the glyoxylate cycle. There is a delay in germination, infection-related development and cuticle penetration in Delta icl1 mutants. Recent reports have shown the importance of the glyoxylate cycle in the virulence of the human pathogenic fungus Candida albicans and the bacterial pathogen Mycobacterium tuberculosis. Our results indicate that the glyoxylate cycle is also important in this plant pathogenic fungus, demonstrating the widespread utility of the pathway in microbial pathogenesis.     

Cellular differentiation and host invasion by the rice blast fungus Magnaporthe grisea

This review describes current advances in understanding the biology of plant infection by the rice blast fungus Magnaporthe grisea. Development of the specialized infection structure, the appressorium, in M. grisea has recently been shown to be controlled by cell cycle progression and initiation of autophagic, programmed cell death in the fungal spore. Re-cycling of the contents of the fungal spore and peroxisomal fatty acid beta-oxidation are therefore important processes for appressorium function. Following entry to the host plant, new evidence suggests that M. grisea grows biotrophically within rice cells, bounded by the plant plasmalemma, and the fungus moves from cell-to-cell by means of plasmodesmata. Biotrophic proliferation of the fungus is likely to require secretion of effector proteins and suppression of host defences. Consistent with this, a component of the polarized exocytosis machinery of M. grisea is necessary for pathogenicity and also for induction of host defences in an incompatible interaction. Large-scale insertional mutagenesis is now allowing the rapid analysis of gene function in M. grisea, heralding a new approach to the study of this important fungal pathogen.     

PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis

The role and function of PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) remains elusive. In this study for the first time, Mtb isogenic mutants missing selected PE_PGRSs were used to investigate their role in the pathogenesis of tuberculosis (TB). We demonstrate that the MtbΔPE_PGRS30 mutant was impaired in its ability to colonize lung tissue and to cause tissue damage, specifically during the chronic steps of infection. Inactivation of PE_PGRS30 resulted in an attenuated phenotype in murine and human macrophages due to the inability of the Mtb mutant to inhibit phagosome–lysosome fusion. Using a series of functional deletion mutants of PE_PGRS30 to complement MtbΔPE_PGRS30, we show that the unique C‐terminal domain of the protein is not required for the full virulence. Interestingly, when Mycobacterium smegmatis recombinant strain expressing PE_PGRS30 was used to infect macrophages or mice in vivo, we observed enhanced cytotoxicity and cell death, and this effect was dependent upon the PGRS domain of the protein.Taken together these results indicate that PE_PGRS30 is necessary for the full virulence of Mtb and sufficient to induce cell death in host cells by the otherwise non‐pathogenic species M. smegmatis, clearly demonstrating that PE_PGRS30 is an Mtb virulence factor.     

A Pmk1-interacting gene is involved in appressorium differentiation and plant infection in Magnaporthe oryzae

In the rice blast fungus Magnaporthe oryzae, the PMK1 mitogen-activated protein (MAP) kinase gene regulates appressorium formation and infectious growth. Its homologs in many other fungi also play critical roles in fungal development and pathogenicity. However, the targets of this important MAP kinase and its interacting genes are not well characterized. In this study, we constructed two yeast two-hybrid libraries of M. oryzae and screened for Pmk1-interacting proteins. Among the nine Pmk1-interacting clones (PICs) identified, two of them, PIC1 and PIC5, were selected for further characterization. Pic1 has one putative nuclear localization signal and one putative MAP kinase phosphorylation site. Pic5 contains one transmembrane domain and two functionally unknown CTNS (cystinosin/ERS1p repeat) motifs. The interaction of Pmk1 with Pic1 or Pic5 was confirmed by coimmunoprecipitation assays. Targeted gene deletion of PIC1 had no apparent effects on vegetative growth and pathogenicity but resulted in a significant reduction in conidiation and abnormal germ tube differentiation on onion epidermal cells. Deletion of PIC5 led to a reduction in conidiation and hyphal growth. Autolysis of aerial hyphae became visible in cultures older than 4 days. The pic5 mutant was defective in germ tube growth and appressorium differentiation. It was reduced in appressorial penetration and virulence on the plant. Both PIC1 and PIC5 are conserved in filamentous ascomycetes, but none of their orthologs have been functionally characterized. Our data indicate that PIC5 is a novel virulence factor involved in appressorium differentiation and pathogenesis in M. oryzae.