A two‐component histidine kinase Shk1 controls stress response,sclerotial formation and fungicide resistance in Sclerotinia sclerotiorum

Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high‐osmolarity glycerol mitogen‐activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H₂O₂‐induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real‐time polymerase chain reaction (qRT‐PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21‐activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild‐type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S. sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two‐component HKs in Sclerotinia.     

Three Fusarium oxysporum mitogen‐activated protein kinases (MAPKs) have distinct and complementary roles in stress adaptation and cross‐kingdom pathogenicity

Mitogen‐activated protein kinase (MAPK) cascades mediate cellular responses to environmental signals. Previous studies in the fungal pathogen Fusarium oxysporum have revealed a crucial role of Fmk1, the MAPK orthologous to Saccharomyces cerevisiae Fus3/Kss1, in vegetative hyphal fusion and plant infection. Here, we genetically dissected the individual and combined contributions of the three MAPKs Fmk1, Mpk1 and Hog1 in the regulation of development, stress response and virulence of F. oxysporum on plant and animal hosts. Mutants lacking Fmk1 or Mpk1 were affected in reactive oxygen species (ROS) homeostasis and impaired in hyphal fusion and aggregation. Loss of Mpk1 also led to increased sensitivity to cell wall and heat stress, which was exacerbated by simultaneous inactivation of Fmk1, suggesting that both MAPKs contribute to cellular adaptation to high temperature, a prerequisite for mammalian pathogens. Deletion of Hog1 caused increased sensitivity to hyperosmotic stress and resulted in partial rescue of the restricted colony growth phenotype of the mpk1Δ mutant. Infection assays on tomato plants and the invertebrate animal host Galleria mellonella revealed distinct and additive contributions of the different MAPKs to virulence. Our results indicate that positive and negative cross‐talk between the three MAPK pathways regulates stress adaptation, development and virulence in the cross‐kingdom pathogen F. oxysporum.     

A two‐component regulatory system VfmIH modulates multiple virulence traits in Dickeya zeae

Bacterial pathogen Dickeya zeae strain EC1 produces antibiotics‐like phytotoxins called zeamines, which are major virulence determinants encoded by the zms gene cluster. In this study, we identified a zeamine‐deficient mutant with a Tn5 insertion in a gene designated as vfmI encoding a two‐component system (TCS) sensor histidine kinase (HK), which is accompanied by vfmH encoding a response regulator (RR) at the same genetic locus. Domain analysis shows this TCS is analogous to the VfmIH of D. dadantii, with typical characteristics of sensor HK and RR, respectively, and sharing the same operon. Deletion of either vfmI or vfmH resulted in decreased production of zeamines and cell wall degrading enzymes (CWDEs), and alleviated virulence on rice seeds and potato tubers. In D. dadantii 3937, VfmH was shown to bind to the promoters of vfmA and vfmE, while in D. zeae EC1, VfmH could bind to the promoters of vfmA, vfmE and vfmF. RNA‐seq analysis of strain EC1 and its vfmH mutant also showed that the TCS positively regulated a range of virulence genes, including zms, T1SS, T2SS, T3SS, T6SS, flagellar and CWDE genes.     

The two‐component systems PrrBA and NtrYX co‐ordinately regulate the adaptation of Brucella abortus to an oxygen‐limited environment

Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world. The success of Brucella as pathogen relies in its ability to adapt to the harsh environmental conditions found in mammalian hosts. One of its main adaptations is the induction of the expression of different genes involved in respiration at low oxygen tension. In this report we describe a regulatory network involved in this adaptation. We show that Brucella abortus PrrBA is a functional two‐component signal transduction system that responds to the redox status and acts as a global regulator controlling the expression of the regulatory proteins NtrY, FnrN and NnrA, which are involved in the adaptation to survive at low oxygen tension. We also show that the two‐component systems PrrBA and NtrYX co‐ordinately regulate the expression of denitrification and high‐affinity cytochrome oxidase genes. Strikingly, a double mutant strain in the prrB and ntrY genes is severely impaired in growth and virulence, while the ntrY and prrB single mutant strains are similar to wild‐type B. abortus. The proposed regulatory network may contribute to understand the mechanisms used by Brucella for a successful adaptation to its replicative niche inside mammalian cells.     

Bacterial ectosymbionts and virulence silencing in a Fusarium oxysporum strain

In the present article we have ascertained the presence of a consortium of ectosymbiotic bacteria belonging to Serratia, Achromobacter, Bacillus and Stenotrophomonas genera associated to the mycelium of the antagonistic Fusarium oxysporum MSA 35 [wild-type (WT) strain]. Morphological characterization carried out on the WT strain, on the F. oxysporum MSA 35 without ectosymbionts [cured (CU) strain] and on the pathogenic F. oxysporum f.sp. lactucae (Fuslat 10) showed that the ectosymbionts, present only in the WT strain, caused a depleted production of micro conidia and aerial hyphae, and a change in shape and dimension of the latter. Virulence tests showed that the cured Fusarium was a pathogenic strain and, as shown by polymerase chain reaction and microscope analysis, pathogenicity was correlated with the capability of the cured hyphae of penetrating lettuce roots. Accordingly, the hyphae of the WT strain were impaired in entering the plant roots. Typing experiments provided evidence that both CU and WT strains belong to F. oxysporum f.sp. lactucae. This implies that the antagonistic effect of WT Fusarium is not a fungal trait, but it is due to the interaction with the ectosymbiotic bacteria. Expression analysis showed that fmk1, chsV and pl1 genes involved in F. oxysporum pathogenicity are not expressed in the WT strain whereas they are expressed in the cured fungus. These results, together with the hyphal characteristics, suggest that the inability of WT strain to penetrate the plant roots could be due to alterations in the expression profile of cell wall-degrading enzymes. In conclusion, we demonstrated a modulation of F. oxysporum gene expression in response to the interaction with the ectosymbiotic bacteria. Preliminary researches indicated that the presence of bacteria attached to the hyphae of antagonistic F. oxysporum is not an isolated phenomenon. Further investigations are necessary to better understand the rule and the diffusion of ectosymbiotic bacteria among antagonistic Fusarium.     

A PR-1-like protein of Fusarium oxysporum functions in virulence on mammalian hosts

The pathogenesis-related PR-1-like protein family comprises secreted proteins from the animal, plant, and fungal kingdoms whose biological function remains poorly understood. Here we have characterized a PR-1-like protein, Fpr1, from Fusarium oxysporum, an ubiquitous fungal pathogen that causes vascular wilt disease on a wide range of plant species and can produce life-threatening infections in immunocompromised humans. Fpr1 is secreted and proteolytically processed by the fungus. The fpr1 gene is required for virulence in a disseminated immunodepressed mouse model, and its function depends on the integrity of the proposed active site of PR-1-like proteins. Fpr1 belongs to a gene family that has expanded in plant pathogenic Sordariomycetes. These results suggest that secreted PR-1-like proteins play important roles in fungal pathogenicity.     

The phosphatase Ptc6 is involved in virulence and MAPK signalling in Fusarium oxysporum

Mitogen-activated kinase (MAPK) signalling pathways are involved in several important processes related to the development and virulence of Fusarium oxysporum. Reversible phosphorylation of the protein members of these pathways is a major regulator of essential biological processes. Among the phosphatases involved in dephosphorylation of MAPKs, type 2C protein phosphatases (PP2Cs) play important roles regulating many developmental strategies and stress responses in yeasts. Nevertheless, the PP2C family is poorly known in filamentous fungi. The F. oxysporum PP2C family includes seven proteins, but only Ptc1 has been studied so far. Here we show the involvement of Ptc6 in the stress response and virulence of F. oxysporum. Expression analysis revealed increased expression of ptc6 in response to cell wall and oxidative stresses. Additionally, targeted inactivation of ptc6 entailed enhanced susceptibility to cell wall stresses caused by Calcofluor White (CFW). We also demonstrate that the lack of Ptc6 deregulates both the Mpk1 phosphorylation induced by CFW and, more importantly, the Fmk1 dephosphorylation induced by pH acidification of the extracellular medium, indicating that Ptc6 is involved in the regulation of these MAPKs. Finally, we showed, for the first time, the involvement of a phosphatase in the invasive growth and virulence of F. oxysporum.     

Rho1 has distinct functions in morphogenesis, cell wall biosynthesis and virulence of Fusarium oxysporum

Rho-type GTPases regulate polarized growth in yeast by reorganization of the actin cytoskeleton and through signalling pathways that control the expression of cell wall biosynthetic genes. We report the cloning and functional analysis of rho1 from Fusarium oxysporum, a soilborne fungal pathogen causing vascular wilt on plants and opportunistic infections in humans. F. oxysporum strains carrying either a Deltarho1 loss-of-function mutation or a rho1(G14V) gain-of-function allele were viable, but displayed a severely restricted colony phenotype which was partially relieved by the osmotic stabilizer sorbitol, indicating structural alterations in the cell wall. Consistent with this hypothesis, Deltarho1 strains showed increased resistance to cell wall-degrading enzymes and staining with Calcofluor white, as well as changes in chitin and glucan synthase gene expression and enzymatic activity. Re-introduction of a functional rho1 allele into the Deltarho1 mutant fully restored the wild-type phenotype. The Deltarho1 strain had dramatically reduced virulence on tomato plants, but was as virulent as the wild type on immunodepressed mice. Thus, Rho1 plays a key role during fungal infection of plants, but not of mammalian hosts.     

Constitutive activation of TORC1 signalling attenuates virulence in the cross-kingdom fungal pathogen Fusarium oxysporum

The filamentous fungus Fusarium oxysporum causes vascular wilt disease in a wide range of plant species and opportunistic infections in humans. Previous work suggested that invasive growth in this pathogen is controlled by environmental cues such as pH and nutrient status. Here we investigated the role of Target Of Rapamycin Complex 1 (TORC1), a global regulator of eukaryotic cell growth and development. Inactivation of the negative regulator Tuberous Sclerosis Complex 2 (Tsc2), but not constitutive activation of the positive regulator Gtr1, in F. oxysporum resulted in inappropriate activation of TORC1 signalling under nutrient-limiting conditions. The tsc2Δ mutants showed reduced colony growth on minimal medium with different nitrogen sources and increased sensitivity to cell wall or high temperature stress. Furthermore, these mutants were impaired in invasive hyphal growth across cellophane membranes and exhibited a marked decrease in virulence, both on tomato plants and on the invertebrate animal host Galleria mellonella. Importantly, invasive hyphal growth in tsc2Δ strains was rescued by rapamycin-mediated inhibition of TORC1. Collectively, these results reveal a key role of TORC1 signalling in the development and pathogenicity of F. oxysporum and suggest new potential targets for controlling fungal infections.     

The F‐box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen‐activated protein kinase (MAPK) pathways in Fusarium oxysporum

F‐box proteins determine substrate specificity of the ubiquitin–proteasome system. Previous work has demonstrated that the F‐box protein Fbp1, a component of the SCF^Fbp1 E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen‐activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum.     

The velvet complex governs mycotoxin production and virulence of Fusarium oxysporum on plant and mammalian hosts

Fungal pathogens provoke devastating losses in agricultural production, contaminate food with mycotoxins and give rise to life‐threatening infections in humans. The soil‐borne ascomycete Fusarium oxysporum attacks over 100 different crops and can cause systemic fusariosis in immunocompromised individuals. Here we functionally characterized VeA, VelB, VelC and LaeA, four components of the velvet protein complex which regulates fungal development and secondary metabolism. Deletion of veA, velB and to a minor extent velC caused a derepression of conidiation as well as alterations in the shape and size of microconidia. VeA and LaeA were required for full virulence of F. oxysporum on tomato plants and on immunodepressed mice. A critical contribution of velvet consists in promoting chromatin accessibility and expression of the biosynthetic gene cluster for beauvericin, a depsipeptide mycotoxin that functions as a virulence determinant. These results reveal a conserved role of the velvet complex during fungal infection on plants and mammals.     

The histidine kinase homologue DhkK/Sombrero controls morphogenesis in Dictyostelium

A key event in Dictyostelium development is the formation of the Mexican hat. This corresponds to a commitment step in morphogenesis that irreversibly signals progression from the slug stage to the fruiting body. We describe the characterization of the dhkK gene that controls this morphogenetic step. Null mutants of dhkK repeatedly attempt, and fail, to undergo morphogenesis from the slug to the Mexican hat, causing them to exhibit a "slugger" phenotype, which cannot be corrected by co-development with wild-type cells. The dhkK gene encodes a putative receptor histidine kinase whose expression is enriched in prestalk cells in the slug. Uniquely for a histidine kinase, DhkK is located in the nuclear envelope. Entry into culmination requires the DhkK response regulator domain, which appears to directly regulate cyclic AMP signaling.     

Fusaric acid contributes to virulence of Fusarium oxysporum on plant and mammalian hosts

Fusaric acid (FA) is amongst the oldest identified secondary metabolites produced by Fusarium species, known for a long time to display strong phytotoxicity and moderate toxicity to animal cells; however, the cellular targets of FA and its function in fungal pathogenicity remain unknown. Here, we investigated the role of FA in Fusarium oxysporum, a soil‐borne cross‐kingdom pathogen that causes vascular wilt on more than 100 plant species and opportunistic infections in humans. Targeted deletion of fub1, encoding a predicted orthologue of the polyketide synthase involved in FA biosynthesis in F. verticillioides and F. fujikuroi, abolished the production of FA and its derivatives in F. oxysporum. We further showed that the expression of fub1 was positively controlled by the master regulator of secondary metabolism LaeA and the alkaline pH regulator PacC through the modulation of chromatin accessibility at the fub1 locus. FA exhibited strong phytotoxicity on tomato plants, which was rescued by the exogenous supply of copper, iron or zinc, suggesting a possible function of FA as a chelating agent of these metal ions. Importantly, the severity of vascular wilt symptoms on tomato plants and the mortality of immunosuppressed mice were significantly reduced in fub1Δ mutants and fully restored in the complemented strains. Collectively, these results provide new insights into the regulation and mode of action of FA, as well as on the function of this phytotoxin during the infection process of F. oxysporum.