Fungal cell wall phosphomannans facilitate the toxic activity of a plant PR-5 protein

Osmotin is a plant PR-5 protein. It has a broad spectrum of antifungal activity, yet also exhibits specificity for certain fungal targets. The structural bases for this specificity remain unknown. We show here that full sensitivity of Saccharomyces cerevisiae cells to the PR-5 protein osmotin is dependent on the function of MNN2, MNN4 and MNN6. MNN2 is an alpha-1, 2-mannosyltransferase catalyzing the addition of the first mannose to the branches on the poly l,6-mannose backbone of the outer chain of cell wall N-linked mannans. MNN4 and MNN6 are required for the transfer of mannosylphosphate to cell wall mannans. Null mnn2, mnn4 or mnn6 mutants lack phosphomannans and are defective in binding osmotin to the fungal cell wall. Both antimannoprotein antibody and the cationic dye alcian blue protect cells against osmotin cytotoxicity. MNN1 is an alpha-1,3-mannosyltransferase that adds the terminal mannose to the outer chain branches of N-linked mannan, masking mannosylphosphate. Null mnn1 cells exhibit enhanced osmotin binding and sensitivity. Several cell wall mannoproteins can bind to immobilized osmotin, suggesting that their polysaccharide constituent determines osmotin binding. Our results demonstrating a causal relationship between cell surface phosphomannan and the susceptibility of a yeast strain to osmotin suggest that cell surface polysaccharides of invading pathogens control target specificity of plant PR-5 proteins.     

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.     

Cell wall integrity: Targeted post-synthetic modifications to reveal its role in plant growth and defense against pathogens

The plant cell wall, a dynamic network of polysaccharides and glycoproteins of significant compositional and structural complexity, functions in plant growth, development and stress responses. In recent years, the existence of plant cell wall integrity (CWI) maintenance mechanisms has been demonstrated, but little is known about the signaling pathways involved, or their components. Examination of key mutants has shed light on the relationships between cell wall remodeling and plant cell responses, indicating a central role for the regulatory network that monitors and controls cell wall performance and integrity. In this review, we present a short overview of cell wall composition and discuss post-synthetic cell wall modification as a valuable approach for studying CWI perception and signaling pathways.     

Extracellular transport and integration of plant secretory proteins into pathogen-induced cell wall compartments

Many fungal parasites enter plant cells by penetrating the host cell wall and, thereafter, differentiate specialized intracellular feeding structures, called haustoria, by invagination of the plant's plasma membrane. Arabidopsis PEN gene products are known to act at the cell periphery and function in the execution of apoplastic immune responses to limit fungal entry. This response underneath fungal contact sites is tightly linked with the deposition of plant cell wall polymers, including PMR4/GSL5-dependent callose, in the paramural space, thereby producing localized wall thickenings called papillae. We show that powdery mildew fungi specifically induce the extracellular transport and entrapment of the fusion protein GFP–PEN1 syntaxin and its interacting partner monomeric yellow fluorescent protein (mYFP)–SNAP33 within the papillary matrix. Remarkably, PMR4/GSL5 callose, GFP–PEN1, mYFP–SNAP33, and the ABC transporter GFP–PEN3 are selectively incorporated into extracellular encasements surrounding haustoria of the powdery mildew Golovinomyces orontii , suggesting that the same secretory defense responses become activated during the formation of papillae and haustorial encasements. This is consistent with a time-course analysis of the encasement process, indicating that these extracellular structures are generated through the extension of papillae. We show that PMR4/GSL5 callose accumulation in papillae and haustorial encasements occurs independently of PEN1 syntaxin. We propose a model in which exosome biogenesis/release serves as a common transport mechanism by which the proteins PEN1 and PEN3, otherwise resident in the plasma membrane, together with membrane lipids, become stably incorporated into both pathogen-induced cell wall compartments.     

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.     

FoEG1, a secreted glycoside hydrolase family 12 protein from Fusarium oxysporum,triggers cell death and modulates plant immunity

Fusarium oxysporum is an important soilborne fungal pathogen with many different formae speciales that can colonize the plant vascular system and cause serious crop wilt disease worldwide. We found a glycoside hydrolase family 12 protein FoEG1, secreted by F. oxysporum, that acted as a pathogen-associated molecular pattern (PAMP) targeting the apoplast of plants to induce cell death. Purified FoEG1 protein triggered cell death in different plants and induced the plant defence response to enhance the disease resistance of plants. The ability of FoEG1 to induce cell death was mediated by leucine-rich repeat (LRR) receptor-like kinases BAK1 and SOBIR1, and this ability was independent of its hydrolase activity. The mutants of cysteine residues did not affect the ability of FoEG1 to induce cell death, and an 86 amino acid fragment from amino acid positions 144 to 229 of FoEG1 was sufficient to induce cell death in Nicotiana benthamiana. In addition, the expression of FoEG1 was strongly induced in the early stage of F. oxysporum infection of host plants, and FoEG1 deletion or loss of enzyme activity reduced the virulence of F. oxysporum. Therefore, our results suggest that FoEG1 can contribute to the virulence of F. oxysporum depending on its enzyme activity and can also act as a PAMP to induce plant defence responses.     

The STRIPAK complex orchestrates cell wall integrity signalling to govern the fungal development and virulence of Fusarium graminearum

Striatin-interacting phosphatases and kinases (STRIPAKs) are evolutionarily conserved supramolecular complexes that control various important cellular processes such as signal transduction and development. However, the role of the STRIPAK complex in pathogenic fungi remains elusive. In this study, the components and function of the STRIPAK complex were investigated in Fusarium graminearum, an important plant-pathogenic fungus. The results obtained from bioinformatic analyses and the protein–protein interactome suggested that the fungal STRIPAK complex consisted of six proteins: Ham2, Ham3, Ham4, PP2Aa, Ppg1, and Mob3. Deletion mutations of individual components of the STRIPAK complex were created, and observed to cause a significant reduction in fungal vegetative growth and sexual development, and dramatically attenuae virulence, excluding the essential gene PP2Aa. Further results revealed that the STRIPAK complex interacted with the mitogen-activated protein kinase Mgv1, a key component in the cell wall integrity pathway, subsequently regulating the phosphorylation level and nuclear accumulation of Mgv1 to control the fungal stress response and virulence. Our results also suggested that the STRIPAK complex was interconnected with the target of rapamycin pathway through Tap42-PP2A cascade. Taken together, our findings revealed that the STRIPAK complex orchestrates cell wall integrity signalling to govern the fungal development and virulence of F. graminearum and highlighted the importance of the STRIPAK complex in fungal virulence.     

The secreted ribonuclease T2 protein FoRnt2 contributes to Fusarium oxysporum virulence

Secreted RNase proteins have been reported from only a few pathogens, and relatively little is known about their biological functions. Fusarium oxysporum is a soilborne fungal pathogen that causes Fusarium wilt, one of the most important diseases on tomato. During the infection of F. oxysporum, some proteins are secreted that modulate host plant immunity and promote pathogen invasion. In this study, we identify an RNase, FoRnt2, from the F. oxysporum secretome that belongs to the ribonuclease T2 family. FoRnt2 possesses an N-terminal signal peptide and can be secreted from F. oxysporum. FoRnt2 exhibited ribonuclease activity and was able to degrade the host plant total RNA in vitro dependent on the active site residues H80 and H142. Deletion of the FoRnt2 gene reduced fungal virulence but had no obvious effect on mycelial growth and conidial production. The expression of FoRnt2 in tomato significantly enhanced plant susceptibility to pathogens. These data indicate that FoRnt2 is an important contributor to the virulence of F. oxysporum, possibly through the degradation of plant RNA.     

Extensin—a major cell wall glycoprotein

Abstract The structure of extensin is described in detail. It has a hydroxyproline-rich backbone, which contains repeating peptides glycosylated by short side chains and it adopts a polyproline II helical conformation. The glycoprotein is synthesized intracellularly and soluble precursors are secreted to the wall, where they are bound, perhaps, by the formation of isodityrosine cross-links. The various hypotheses, including the most recent ‘warp and weft’ model, which have been suggested to explain the attachment of extensin to the other wall polymers are discussed. The possible functions of extensin in defence and in the control of extension growth are described in addition to its probable structural role. Other glycoproteins which resemble extensin are also mentioned.     

Polygalacturonase-inhibiting protein is a structural component of plant cell wall

It is generally believed that plants "evolved a strategy of defending themselves from a phytopathogen attack" during evolution. This metaphor is used frequently, but it does not facilitate understanding of the mechanisms providing plant resistance to the invasion of foreign organisms and to other unfavorable external factors, as well as the role of these mechanisms in plant growth and development. Information on processes involving one of the plant resistance factors--polygalacturonase-inhibiting protein (PGIP)--is considered in this review. The data presented here indicate that PGIP, being an extracellular leucine-rich repeat-containing protein, performs important functions in the structure of plant cell wall. Amino acid residues participating in PGIP binding to homogalacturonan in the cell wall have been determined. The degree of methylation and the mode of distribution of homogalacturonan methyl groups are responsible for the formation of a complex structure, which perhaps determines the specificity of PGIP binding to pectin. PGIP is apparently one of the components of plant cell wall determining some of its mechanical properties; it is involved in biochemical processes related to growth, expansion, and maceration, and it influences plant morphology. Polygalacturonase (PG) is present within practically all plant tissues, but the manifestation of its activity varies significantly depending on physiological conditions in the tissue. Apparently, the regulation of PG functioning in apoplast significantly affects the development of processes associated with the modification of the structure of plant cell wall. PGIP can regulate PG activity through binding to homogalacturonan. The genetically determined structure of PGIP in plants determines the mode of its interaction with an invader and perhaps is one of the factors responsible for the set of pathogens causing diseases in a given plant species.     

Molecular defense responses in roots and the rhizosphere against Fusarium oxysporum

Plants face many different concurrent and consecutive abiotic and biotic stresses during their lifetime. Roots can be infected by numerous pathogens and parasitic organisms. Unlike foliar pathogens, root pathogens have not been explored enough to fully understand root-pathogen interactions and the underlying mechanism of defense and resistance. PR gene expression, structural responses, secondary metabolite and root exudate production, as well as the recruitment of plant defense–assisting “soldier” rhizosphere microbes all assist in root defense against pathogens and herbivores. With new high-throughput molecular tools becoming available and more affordable, now is the opportune time to take a deep look below the ground. In this addendum, we focus on soil-borne Fusarium oxysporum as a pathogen and the options plants have to defend themselves against these hard-to-control pathogens.     

Heterotrimeric G-proteins of a filamentous fungus regulate cell wall composition and susceptibility to a plant PR-5 protein

Membrane permeabilizing plant defensive proteins first encounter the fungal cell wall that can harbor specific components that facilitate or prevent access to the plasma membrane. However, signal transduction pathways controlling cell wall composition in filamentous fungi are largely unknown. We report here that the deposition of cell wall constituents that block the action of osmotin (PR-5), an antifungal plant defense protein, against Aspergillus nidulans requires the activity of a heterotrimeric G-protein mediated signaling pathway. The guanidine nucleotide GDPbetaS, that locks G-proteins in a GDP-bound inactive form, inhibits osmotin-induced conidial lysis. A dominant interfering mutation in FadA, the alpha-subunit of a heterotrimeric G-protein, confers resistance to osmotin. A deletion mutation in SfaD, the beta-subunit of a heterotrimeric G-protein also increases osmotin resistance. Aspergillus nidulans strains bearing these mutations also have increased tolerance to SDS, reduced cell wall porosity and increased chitin content in the cell wall.     

The state of cell wall pectin monitored by wall associated kinases: A model

The Wall Associated Kinases (WAKs) bind to both cross-linked polymers of pectin in the plant cell wall, but have a higher affinity for smaller fragmented pectins that are generated upon pathogen attack or wounding. WAKs are required for cell expansion during normal seedling development and this involves pectin binding and a signal transduction pathway involving MPK3 and invertase induction. Alternatively WAKs bind pathogen generated pectin fragments to activate a distinct MPK6 dependent stress response. Evidence is provided for a model for how newly generated pectin fragments compete for longer pectins to alter the WAK dependent responses.     

Effect of a Bacillus subtilis strain on flax protection against Fusarium oxysporum and its impact on the root and stem cell walls

In Normandy, flax is a plant of important economic interest because of its fibres. Fusarium oxysporum, a telluric fungus, is responsible for the major losses in crop yield and fibre quality. Several methods are currently used to limit the use of phytochemicals on crops. One of them is the use of plant growth promoting rhizobacteria (PGPR) occurring naturally in the rhizosphere. PGPR are known to act as local antagonists to soil‐borne pathogens and to enhance plant resistance by eliciting the induced systemic resistance (ISR). In this study, we first investigated the cell wall modifications occurring in roots and stems after inoculation with the fungus in two flax varieties. First, we showed that both varieties displayed different cell wall organization and that rapid modifications occurred in roots and stems after inoculation. Then, we demonstrated the efficiency of a Bacillus subtilis strain to limit Fusarium wilt on both varieties with a better efficiency for one of them. Finally, thermo‐gravimetry was used to highlight that B. subtilis induced modifications of the stem properties, supporting a reinforcement of the cell walls. Our findings suggest that the efficiency and the mode of action of the PGPR B. subtilis is likely to be flax variety dependent.     

The transmembrane protein Sho1 cooperates with the mucin Msb2 to regulate invasive growth and plant infection in Fusarium oxysporum

In the vascular wilt pathogen Fusarium oxysporum, the mitogen‐activated protein kinase (MAPK) Fmk1 is essential for plant infection. The mucin‐like membrane protein Msb2 regulates a subset of Fmk1‐dependent functions. Here, we examined the role of the tetraspan transmembrane protein Sho1 as an additional regulator of the Fmk1 pathway and determined its genetic interaction with Msb2. Targeted Δsho1 mutants were generated in wild‐type and Δmsb2 backgrounds to test possible interactions between the two genes. The mutants were examined for hyphal growth under different stress conditions, phosphorylation of the MAPK Fmk1 and an array of Fmk1‐dependent virulence functions. Similar to Msb2, Sho1 was required for the activation of Fmk1 phosphorylation, as well as Fmk1‐dependent gene expression and invasive growth functions, including extracellular pectinolytic activity, cellophane penetration, plant tissue colonization and virulence on tomato plants. Δsho1 mutants were hypersensitive to the cell wall‐perturbing compound Calcofluor White, and this phenotype was exacerbated in the Δmsb2 Δsho1 double mutant. These results highlight that Sho1 and Msb2 have partially overlapping functions upstream of the Fmk1 MAPK cascade, to promote invasive growth and plant infection, as well as cell wall integrity, in F. oxysporum.     

Do microtubules orient plant cell wall microfibrils?

Cortical microtubules (MTs) allegedly orient nascent cellulose microfibrils (CMFs) in plant cells. The frequently observed parallelism between them, and the effect of MT-depolymerizing agents, are the bases for this hypothesis. Data have, however, accumulated about cells in which MTs and CMFs are not in parallel alignment. These data will be reviewed. MT orientation cannot be the only factor determining CMF orientation, but MTs could overrule other factors in cells where, for instance, they are more tightly attached to the plasma membrane than in other cells. MT and CMF orientations could, however, both be controlled by a third factor, and CMFs may even impose orientation on MTs.     

An update on receptor-like kinase involvement in the maintenance of plant cell wall integrity

Background

Plant cell walls form the interface between the cells and their environment. They perform different functions, such as protecting cells from biotic and abiotic stress and providing structural support during development. Maintenance of the functional integrity of cell walls during these different processes is a prerequisite that enables the walls to perform their particular functions. The available evidence suggests that an integrity maintenance mechanism exists in plants that is capable of both detecting wall integrity impairment caused by cell wall damage and initiating compensatory responses to maintain functional integrity. The responses involve 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonic acid, reactive oxygen species and calcium-based signal transduction cascades as well as the production of lignin and other cell wall components. Experimental evidence implicates clearly different signalling molecules, but knowledge regarding contributions of receptor-like kinases to this process is less clear. Different receptor-like kinase families have been considered as possible sensors for perception of cell wall damage; however, strong experimental evidence that provides insights into functioning exists for very few kinases.

Scope and Conclusions

This review examines the involvement of cell wall integrity maintenance in different biological processes, defines what constitutes plant cell wall damage that impairs functional integrity, clarifies which stimulus perception and signal transduction mechanisms are required for integrity maintenance and assesses the available evidence regarding the functions of receptor-like kinases during cell wall integrity maintenance. The review concludes by discussing how the plant cell wall integrity maintenance mechanism could form an essential component of biotic stress responses and of plant development, functions that have not been fully recognized to date.