MoMon1 is required for vacuolar assembly,conidiogenesis and pathogenicity in the rice blast fungus Magnaporthe oryzae

Mon1 protein is involved in cytoplasm-to-vacuole trafficking, vacuolar morphology and autophagy, and is required for homotypic vacuole fusion in Saccharomyces cerevisiae. Here we identify MoMON1 from Magnaporthe oryzae as an ortholog of S. cerevisiae MON1, essential for the morphology of the vacuole and vesicle fusion. Target gene deletion of MoMON1 resulted in accumulation of small punctuate vacuoles in the hypha and hypersensitivity to monensin, an antibiotic that blocks intracellular protein transport. The ΔMomon1 mutant exhibited significantly reduced aerial hyphal development and poor conidiation. Conidia of ΔMomon1 were able to differentiate appressoria. However, ΔMomon1 was non-pathogenic on rice leaves, even after wound inoculation. In addition, ΔMomon1 was slightly hypersensitive to Congo red and SDS, but not to cell wall degrading enzymes, suggesting significant alterations in its cell wall. The autophagy process was blocked in the ΔMomon1 mutant. Taken together, our results suggest that MoMON1 has an essential function in vacuolar assembly, autophagy, fungal development and pathogenicity in M. oryzae.     

Transformation of the rice blast fungus Magnaporthe grisea to hygromycin B resistance

Summary Low frequency, integrative transformation of three fertile hermaphroditic strains of Magnaporthe grisea has been achieved using plasmid pAN7-1 and cosmid pAN7-2, which contain an Escherichia coli hygromycin B phosphotransferase gene linked to Aspergillus nidulans regulatory sequences.     

A spindle pole antigen gene MoSPA2 is important for polar cell growth of vegetative hyphae and conidia,but is dispensable for pathogenicity in Magnaporthe oryzae

Spa2 is an important component of the multiprotein complex polarisome, which is involved in the establishment, maintenance, termination of polarized cell growth and is important for defining tip growth of filamentous fungi. In this study, we isolated an insertional mutant of the rice blast fungus Magnaporthe oryzae that formed smaller colony and conidia compared with the wild type. In the mutant, a spindle pole antigen gene MoSPA2 was disrupted by the integration of an exogenous plasmid. Targeted gene deletion and complementation assays demonstrated the gene disruption was responsible for the defects of the insertional mutant. Interestingly, the MoSpa2-GFP fusion protein was found to accumulate as a spot at hyphal tips, septa of hyphae and conidial tip cells where germ tubes are usually produced, but not in appressoria, infection hyphae or at the septa of conidia. Furthermore, the deletion mutants of MoSPA2 exhibited slower hyphal tip growth, more hyphal branches, and smaller size of conidial tip cells. However, MoSPA2 is not required for plant infection. These results indicate that MoSPA2 is required for vegetative hyphal growth and maintaining conidium morphology and that spotted accumulation of MoSpa2 is important for its functions during cell polar growth.     

Organisation and molecular analysis of repeated DNA sequences in the rice blast fungus Magnaporthe grisea

The distribution of a previously described repeated DNA sequence present as a 1.3-kb PstI fragment in the genome of the rice blast fungus Magnaporthe grisea was analysed by carrying out DNA fingerprint analysis of 36 isolates including rice, non-rice and laboratory strains. The analysis of various higher-molecular-weight PstI fragments with homology to the 1.3-kb repeat revealed that these may arise predominantly from transposon insertions or point mutations. Analysis of a 5.1-kb derivative revealed both a point mutation at a PstI site and an insertion of a putative transposable element which caused an increase in molecular weight from 1.3 to 5.1 kb. Another repeat element of 1.4 kb was identified and found to exist in association with the 1.3-kb repeat. Both 1.3- and 1.4-kb elements were found to be parts of MGR583 (Hamer et al. 1989), a LINE-like element. These elements were present in a high copy number in all the rice and a majority of non-rice pathogens indicating that MGR583 is not a host-specific sequence as reported earlier. Our results suggest that repeated DNA elements in M. grisea have amplified independently of one another and further indicate that different isolates of M. grisea may have evolved from several distinct lines of origin. Received: 12 April / 12 November 1996     

Mycobacterium marinum Erp is a virulence determinant required for cell wall integrity and intracellular survival

The Mycobacterium tuberculosis exported repetitive protein (Erp) is a virulence determinant required for growth in cultured macrophages and in vivo. To better understand the role of Erp in Mycobacterium pathogenesis, we generated a mutation in the erp homologue of Mycobacterium marinum, a close genetic relative of M. tuberculosis. erp-deficient M. marinum was growth attenuated in cultured macrophage monolayers and during chronic granulomatous infection of leopard frogs, suggesting that Erp function is similarly required for the virulence of both M. tuberculosis and M. marinum. To pinpoint the step in infection at which Erp is required, we utilized a zebrafish embryo infection model that allows M. marinum infections to be visualized in real-time, comparing the erp-deficient strain to a DeltaRD1 mutant whose stage of attenuation was previously characterized in zebrafish embryos. A detailed microscopic examination of infected embryos revealed that bacteria lacking Erp were compromised very early in infection, failing to grow and/or survive upon phagocytosis by host macrophages. In contrast, DeltaRD1 mutant bacteria grow normally in macrophages but fail to induce host macrophage aggregation and subsequent cell-to-cell spread. Consistent with these in vivo findings, erp-deficient but not RD1-deficient bacteria exhibited permeability defects in vitro, which may be responsible for their specific failure to survive in host macrophages.     

The P-type ATPase Spf1 is required for endoplasmic reticulum functions and cell wall integrity in Candida albicans

Endoplasmic reticulum (ER) is crucial for protein folding, glycosylation and secretion in eukaryotic organisms. These important functions are supported by high levels of Ca²⁺ in the ER. We have recently identified a putative ER Ca²⁺ pump in Candida albicans, called Spf1, which plays key roles in maintenance of cellular Ca²⁺ homeostasis, morphogenesis and virulence. In this study, we purified Spf1 and confirmed that it is a P-type ATPase, suggesting its role in maintaining high levels of ER Ca²⁺. Disruption of SPF1 caused severe defects in glycosylation of the ER-localized protein Cdc101 and secretory acid phosphatase, and a decrease in expression of SEC61 which encodes an important ER protein. Moreover, the spf1Δ/Δ mutant showed increased sensitivity to cell wall stresses, abnormal cell wall composition, delayed cell wall reconstruction and decreased flocculation and adherence, indicating its defect in cell wall integrity (CWI). We also revealed that disruption of SPF1 has an impact on gene expression related to CWI and morphogenesis. This study provides evidence that Spf1, as a P-type ATPase, is essential for ER functions and consequent CWI, implicating a role of ER Ca²⁺ homeostasis in C. albicans physiology.     

Identification of three ubiquitin genes of the rice blast fungus Magnaporthe grisea, one of which is highly expressed during initial stages of plant colonisation

Differential cDNA screening was used to identify genes expressed during the colonisation of rice leaves by the pathogenic fungus Magnaporthe grisea. This led to the identification of a gene, called UEP1, which encodes a ubiquitin extension protein. UEP1 was highly expressed 48 h after initial fungal infection of rice leaves when M. grisea is proliferating in the leaf epidermis but not yet causing disease symptoms. UEP1 appeared to be down-regulated after this time despite further extensive growth of the fungus throughout the leaf tissue. To investigate the potential role of ubiquitin in fungal pathogenesis we subsequently isolated UEP3 and PUB4, encoding a second ubiquitin extension protein and a polyubiquitin respectively. UEP1 was expressed abundantly during active growth of M. grisea in axenic culture but was down-regulated by starvation-stress. UEP3 showed a similar pattern of expression to UEP1 during the growth of M. grisea in culture and after environmental stress, but was not highly expressed during plant colonisation. PUB4 was highly expressed after environmental stress, but was not highly expressed during plant colonisation. UEP1 was found to be present in a much-higher copy number per haploid genome compared to UEP3 and PUB4. The restricted high-level expression of UEP1 suggests that M. grisea undergoes rapid ribosomal biogenesis and protein turnover during initial plant-tissue colonisation, which is regulated by a specific UEP1-encoded component of the M. grisea ubiquitin gene family. Received: 23 December 1997 / 17 March 1998     

Ksg1, a homologue of the phosphoinositide-dependent protein kinase 1, controls cell wall integrity in Schizosaccharomyces pombe

It has previously been shown that the Schizosaccharomyces pombe mutant ksg1-358 has a mating and sporulation defect at 30 degrees C and that it is temperature sensitive for growth at 35 degrees C. However the molecular basis for these phenotypes remained largely unknown. In this study we show that ksg1-358 mutant cells lysed at the non-permissive temperature, which could be prevented by sorbitol. Overexpression of ksg1 using the nmt1-promoter showed slow growth and cells became swollen when incubated at 35 degrees C under low inositol conditions. Interestingly, in a two-hybrid assay we found that the ksg1-protein interacted with Pck1p, a protein implicated in regulating cell wall integrity in S. pombe. Genetic complementation assays showed that an overexpression of pck2, the homologue of pck1 involved in the regulation of cell wall synthesis, could partially rescue ksg1-358 phenotypes. We digested the ksg1-358 cell wall using beta-glucanase. We found that the ksg1-358 mutant was more resistant to cell lysis at 30 degrees C than the wildtype strain h972, which was similar to a pck1-deletion strain. A ksg1-overexpressing strain was hypersensitive towards beta-glucanase treatment similar to a pck2-deletion strain. The pck1-deletion partially rescued beta-glucanase hypersensitivity of the ksg1-overexpressing strain but the pck2-deletion increased it. The ksg1-358 mutation increased beta-glucanase resistance of a pck1-overexpressing strain but it had no effect on a pck2-overexpressing strain. Our results provide evidence that ksg1 is a novel regulator of cell wall integrity in the fission yeast Schizosaccharomyces pombe. They further suggest that Ksg1p acts in a pathway with Pck1p, possibly upstream and through direct interaction.     

AfMkk2 is required for cell wall integrity signaling,adhesion, and full virulence of the human pathogen Aspergillus fumigatus

The cell wall integrity (CWI) pathway, best characterized in S. cerevisiae, is strikingly conserved in Aspergillus species. We analyzed the importance of AfMkk2, a CWI signaling kinase, for virulence and antifungal therapy in the human pathogen A. fumigatus. A mutant lacking AfMkk2 is less adherent to glass and plastic surfaces and shows increased sensitivity to alkaline pH stress and antifungals. Rather than AfMpkA, the target kinase of AfMkk2, AfMpkB is activated in the mutant under cell wall stress. Interestingly, the mutant lacking AfMkk2 shows an enhanced sensitivity to posaconazole and voriconazole. And in agreement with its sensitivity to moderate temperatures, it is less virulent in a murine infection model. Our data underline the importance of mkk2 for the fitness, but also for the pathogenicity of A. fumigatus.     

COS-l, a putative two-component histidine kinase of Candida albicans, is an in vivo virulence factor.

The human fungal pathogen, Candida albicans, has three putative histidine kinases showing homology to those of plants, bacteria and other fungi. We have constructed a homozygous deletion strain and a hemizygous reconstituted strain of one of these histidine-kinase-encoding genes, COS-1, in C. albicans. Neither strain showed any growth defect in a number of liquid media nor increased resistance or sensitivity to a number of antifungal drugs. Importantly, we show that the COS-1 homozygous disruption strain had significantly reduced virulence in a systemic murine model of candidosis. Thus, COS-1 appears to be an in vivo virulence factor and may represent a novel target for the development of antifungal drugs.     

<Emphasis Type="Italic">MoDUO1</Emphasis>, a Duo1-like gene,is required for full virulence of the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Duo1, a major component of the Dam1 complex which has been found in two species of yeast (the budding yeast Saccharomyces cerevisae and the fission yeast Schizosaccharomyces pombe), is involved in mitosis-related chromosome segregation, while its relevance to pathogenicity in filamentous fungi remains unclear. This report elucidated this very fact in the case of the rice blast fungus Magnaporthe oryzae. A gene designated MoDUO1 that encodes a Duo1-like homolog (MoDuo1) was discovered in the M. oryzae genome. Two types of MoDUO1 mutants were obtained using genetic approaches of Agrobacterium-mediated gene disruption and homologous recombination. Both disruption and deletion of MoDUO1 can exert profound effects on the formation pattern of conidiophores and conidial morphology, such as abnormal nucleic numbers in conidia and delayed extension of infectious hyphae. Intriguingly, plant infection assays demonstrated that inactivation of MoDUO1 significantly attenuates the virulence in its natural host rice leaves, and functional complementation can restore it. Subcellular localization assays showed that MoDuo1 is mainly distributed in the cytosol of fungal cells. Proteomics-based investigation revealed that the expression of four mitosis-related proteins is shut down in the MoDUO1 mutant, suggesting that MoDuo1 may have a function in mitosis. In light of the fact that Duo1 orthologs are widespread in plant and human fungal pathogens, our finding may represent a common mechanism underlying fungal virulence. To the best of our knowledge, this is the first example of linking a Duo1-like homolog to the pathogenesis of a pathogenic fungus, which might provide clues to additional studies on the role of Dam1 complex in M. oryzae and its interaction with rice.     

Involvement of putative response regulator genes of the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in osmotic stress response,fungicide action,and pathogenicity

Rice blast fungus (Magnaporthe oryzae) has ten histidine kinases (HKs), one histidine-containing phosphotransfer protein (HPt), and three response regulators (RRs) as putative components of the two-component signal transduction system (TCS). Here, we constructed knockout mutants of two putative RR genes (MoSSK1, MoSKN7) and a RR homolog gene (MoRIM15) to analyze the roles of TCS in environmental adaptation and pathogenicity. The DeltaMossk1 strain had increased sensitivity to high osmolarity and decreased sensitivity to fludioxonil. The DeltaMoskn7 strain had slightly decreased sensitivity to fludioxonil. The involvement of MoSkn7 in the osmoresponse was obvious only on the DeltaMossk1 background. These results show that MoSsk1 and MoSkn7 are major and minor contributors, respectively, in the high osmolarity response and fludioxonil action. The DeltaMossk1 strain was more osmosensitive than the predicted upstream HK gene disruptant Deltahik1, which shows sugar-specific high osmolarity sensitivity. The DeltaMossk1 and DeltaMoskn7 strains showed enhanced hyphal melanization, suggesting that RRs regulate hyphal melanization. MoSsk1 and MoRim15 are required for full virulence, because the DeltaMossk1 and DeltaMorim15 strains exhibited reduced virulence. These results suggest that the putative RRs of the rice blast fungus are involved in the osmotic stress response, fludioxonil action, and pathogenicity.     

The hybrid histidine kinase Hk1 is part of a two-component system that is essential for survival of Borrelia burgdorferi in feeding Ixodes scapularis ticks

Two-component systems (TCS) are principal mechanisms by which bacteria adapt to their surroundings. Borrelia burgdorferi encodes only two TCS. One is comprised of a histidine kinase, Hk2, and the response regulator Rrp2. While the contribution of Hk2 remains unclear, Rrp2 is part of a regulatory pathway involving the spirochete's alternate sigma factors, RpoN and RpoS. Genes within the Rrp2/RpoN/RpoS regulon function to promote tick transmission and early infection. The other TCS consists of a hybrid histidine kinase, Hk1, and the response regulator Rrp1. Hk1 is composed of two periplasmic sensor domains (D1 and D2), followed by conserved cytoplasmic histidine kinase core, REC, and Hpt domains. In addition to its REC domain, Rrp1 contains a GGDEF motif characteristic of diguanylate cyclases. To investigate the role of Hk1 during the enzootic cycle, we inactivated this gene in two virulent backgrounds. Extensive characterization of the resulting mutants revealed a dramatic phenotype whereby Hk1-deficient spirochetes are virulent in mice and able to migrate out of the bite site during feeding but are killed within the midgut following acquisition. We hypothesize that the phosphorelay between Hk1 and Rrp1 is initiated by the binding of feeding-specific ligand(s) to Hk1 sensor domain D1 and/or D2. Once activated, Rrp1 directs the synthesis of cyclic dimeric GMP (c-di-GMP), which, in turn, modulates the expression and/or activity of gene products required for survival within feeding ticks. In contrast to the Rrp2/RpoN/RpoS pathway, which is active only within feeding nymphs, the Hk1/Rrp1 TCS is essential for survival during both larval and nymphal blood meals.     

Drosophila glob1 is required for the maintenance of cytoskeletal integrity during oogenesis

Background: Hemoglobins (Hbs) are evolutionarily conserved heme‐containing metallo‐proteins of the Globin protein family that harbour the characteristic “globin fold.” Hemoglobins have been functionally diversified during evolution and their usual property of oxygen transport is rather a recent adaptation. Drosophila genome possesses three globin genes (glob1, glob2, and glob3), and we have reported earlier that adequate expression of glob1 is required for various aspects of development, as well as to regulate the cellular level of reactive oxygen species (ROS). The present study illustrates the explicit role of Drosophila globin1 in progression of oogenesis. Results: We demonstrate a dynamic expression pattern of glob1 in somatic and germ cell derivatives of developing egg chambers during various stages of oogenesis, which largely confines around the F‐actin‐rich cellular components. Reduced expression of glob1 leads to various types of abnormalities during oogenesis, which were primarily mediated by the inappropriately formed F‐actin‐based cytoskeleton. Our subsequent analysis in the somatic and germ line clones shows cell autonomous role of glob1 in the maintenance of the integrity of F‐actin‐based cytoskeleton components in the somatic and germ cell derivatives. Conclusions: Our study establishes a novel role of glob1 in maintenance of F‐actin‐based cytoskeleton during progression of oogenesis in Drosophila. Developmental Dynamics 245:1048–1065, 2016. © 2016 Wiley Periodicals, Inc.     

A 60-kilodalton immunodominant glycoprotein is essential for cell wall integrity and the maintenance of cell shape in Streptococcus mutans

We have demonstrated previously by Western blotting that in naturally sensitized humans, the serum or salivary antibody response to Streptococcus mutans was directed predominantly to a protein antigen with a size of approximately 60-kDa. To identify this immunodominant antigen, specific serum antibodies were eluted from immunoblots and five positive clones with inserts ranging in length from 3 to 8 kb from identical chromosomal loci were obtained by screening a genomic expression library of Streptococcus mutans GS-5. Amino acid sequencing established the identity of this immunodominant antigen, a 60-kDa immunodominant glycoprotein (IDG-60), to be a cell wall-associated general stress protein GSP-781, which was originally predicted to have a molecular mass of approximately 45 kDa based on the derived nucleotide sequence. Discrepancy in the molecular mass was also observed in recombinant his-tagged IDG-60 (rIDG-60) expressed from Escherichia coli. Glycosylation, consisting of sialic acid, mannose galactose, and N-acetylgalactosamine, was detected by lectin binding to IDG-60 in cell wall extracts from S. mutans and rIDG-60 expressed in vivo or translated in vitro. Despite the presence of multiple Asn or Ser or Thr glycosylation sites, IDG-60 was resistant to the effect of N-glycosidase F and multiple O-glycosidase molecules but not to beta-galactosidase. Insertional inactivation of the gene encoding IDG-60, sagA, resulted in a retarded growth rate, destabilization of the cell wall, and pleiomorphic cell shape with multifold ingrowth of cell wall. In addition, distinct from the parental GS-5 strain, the isogenic mutant GS-51 was unable to survive the challenge of low pH and high osmotic pressure or high temperature. Expression of the wild-type gene in trans within GS-51 from plasmid pDL277 complemented the growth defect and restored normal cell shape. These results suggested that IDG-60 is essential for maintaining the integrity of the cell wall and the uniformity of cell shape, both of which are indispensable for bacteria survival under stress conditions.