Specialized Infection Structures Research Articles

The phosphorylation landscape of infection-related development by the rice blast fungus

Many of the world's most devastating crop diseases are caused by fungal pathogens that elaborate specialized infection structures to invade plant tissue. Here, we present a quantitative mass-spectrometry-based phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae, which threatens global food security. We mapped 8,005 phosphosites on 2,062 fungal proteins following germination on a hydrophobic surface, revealing major re-wiring of phosphorylation-based signaling cascades during appressorium development. Comparing phosphosite conservation across 41 fungal species reveals phosphorylation signatures specifically associated with biotrophic and hemibiotrophic fungal infection. We then used parallel reaction monitoring (PRM) to identify phosphoproteins regulated by the fungal Pmk1 MAPK that controls plant infection by M.oryzae. We define 32 substrates of Pmk1 and show that Pmk1-dependent phosphorylation of regulator Vts1 is required for rice blast disease. Defining the phosphorylation landscape of infection therefore identifies potential therapeutic interventions for the control of plant diseases.

Peroxin MoPex22 Regulates the Import of Peroxisomal Matrix Proteins and Appressorium-Mediated Plant Infection in Magnaporthe oryzae.

Magnaporthe oryzae, the pathogen responsible for rice blast disease, utilizes specialized infection structures known as appressoria to breach the leaf cuticle and establish intracellular, infectious hyphae. Our study demonstrates that the peroxin MoPex22 is crucial for appressorium function, specifically for the development of primary penetration hyphae. The ∆Mopex22 mutant exhibited slow growth, reduced aerial hyphae, and almost complete loss of virulence. Specifically, despite the mutant's capability to form appressoria, it showed abnormalities during appressorium development, including reduced turgor, increased permeability of the appressorium wall, failure to form septin rings, and significantly decreased ability to penetrate host cells. Additionally, there was a delay in the degradation of lipid droplets during conidial germination and appressorium development. Consistent with these findings, the ΔMopex22 mutant showed an inefficient utilization of long-chain fatty acids and defects in cell wall integrity. Moreover, our findings indicate that MoPex22 acts as an anchor for MoPex4, facilitating the localization of MoPex4 to peroxisomes. Together with MoPex4, it affects the function of MoPex5, thus regulating the import of peroxisomal matrix proteins. Overall, these results highlight the essential role of MoPex22 in regulating the transport of peroxisomal matrix proteins, which affect fatty acid metabolism, glycerol accumulation, cell wall integrity, growth, appressorium development, and the pathogenicity of M. oryzae. This study provides valuable insights into the significance of peroxin functions in fungal biology and appressorium-mediated plant infection.

The low-affinity calcium channel protein AaFig1 is essential for the growth and development, infection structure differentiation, and pathogenicity of Alternaria alternata

Fig1, as a low-affinity Ca2+ channel, is needed for Ca2+ influx and normal mating found in a few species, but its detailed molecular regulating mechanism in fungi, especially in Alternaria alternata, the causal agent of pear black spot, is still unclear. In this study, Fig1 in A. alternata was cloned, identified and named as AaFig1, and its function was characterized by targeted gene knockout strategy. The results showed that the ΔAaFig1 was severely decreased in vegetative growth, biomass accumulation and sporulation. Fluo-4, AM staining indicated that the Ca2+ content in ΔAaFig1 was decreased. Deletion of AaFig1 reduced the response of A. alternata to exogenous Ca2+ but improved the tolerance to osmotic stresses. Interestingly, the appressorium formation of the ΔAaFig1 strain on hydrophobic and pear wax extract coated surface, and infection hyphae formation on the onion epidermis were significantly reduced, meanwhile, the ΔAaFig1 strain could not penetrate the cellophane. In addition, virulence on wound-inoculated pear fruit and intact pear leaves, melanin production and the activities of cell wall degrading enzymes in AaFig1 strain were reduced. In summary, these results indicate that AaFig1-mediated Ca2+ homeostasis positively regulates the growth and development, infection structure formation and pathogenicity of A. alternata. The findings will provide a basis for developing new strategies to control this pathogen in pear fruit.

The sensor protein AaSln1 is involved in differentiation of infection structures, osmotic stress tolerance and virulence in Alternaria alternata

Sln, a conserved sensor kinase, plays a distinct function in different fungi. The gene AaSln1 in Alternaria alternata was successfully cloned and characterized. Sequence analysis revealed that AaSln1 consists of a transmembrane helix region, two characteristic low complexity regions, and a HisKA, HATPase_c and sesponse reg domain, respectively. Through gene knockout strategy, the results showed that AaSln1 significantly regulated infection structural differentiation, pathogenicity, stress response and melanin synthesis and secretion, while had no effect on growth, sporulation and biomass accumulation of A. alternata. The AaSln1 regulates pathways in A. alternata including ABC transporters, starch and sucrose metabolism according to the transcriptomic analysis. RNA-Seq results revealed that the gene expression levels of the downstream regulators of Hog1-MAPK pathway such as Mss4, Ste12, Fsk2 and Mip1 were significantly reduced in the ΔAaSln1 mutant. These findings demonstrated that AaSln1 has an essential role in the pathogenicity and regulates downstream biological processes of A. alternata by activating the MAPK pathway.

Spermidine synthase is essential for vegetative growth, stress response, secondary metabolism and pathogenicity in Alternaria alternata

Black spot, caused by the fungus Alternaria alternata, is one of the postharvest diseases that cause significant economic losses. Polyamine metabolism in fungi plays key roles in cellular processes and pathogen-host interactions. However, little is known about their detailed regulatory mechanisms on pathogenicity of plant fungi. In this study, we characterized the spermidine synthase AaSPDS in A. alternata. Knocking out AaSPDS caused a total loss of vegetative growth and sporulation which was partially restored by exogenous spermidine (Spd), so supplying 0.5 mM exogenous Spd to medium for evaluating the characteristics of WT and ΔAaSPDS. The deletion strain showed albino colonies, sparse mycelial tips and significant decreased biomass accumulation and melanin production. ΔAaSPDS strain was also more sensitive to osmotic stress, oxidative stress and cell wall-perturbing agents. Interestingly, the expression level of AaSPDS was greatly up-regulated during infection structure differentiation stage of A. alternata, and appressorium formation of the AaSPDS deletion strain was completely blocked. In addition, deletion of the AaSPDS gene led to a significant reduction in pathogenicity to pear fruit, altenuene (ALT) mycotoxin accumulation and the activities of cellulase (CL), β-glucosidase (β-GC) and polygalacturonase (PG). The results presented in this study suggested that AaSPDS influences the pathogenicity of A. alternata by regulating infection structure formation, cell wall degrading enzyme (CWDE) activity and mycotoxin production.

Distinct dynamics of the nucleolus in response to nutrient availability and during development in the rice blast fungus.

The nucleolus is a dynamic subnuclear structure that is involved in many fundamental processes of the nucleus. In higher eukaryotic cells, the size and shape of nucleoli correlate with nucleolar activities. For fungi, knowledge of the nucleolus and its functions is primarily gleaned from budding yeast. Whether such correlation is conserved and how nucleolar functions are regulated in filamentous fungi including important human and crop pathogens are largely unknown. Our observations reveal that the dynamics of nucleolus in a model plant pathogenic fungus, Magnaporthe oryzae, is distinct from those of animal and yeast nucleoli under low nutrient availability and during pathogenic development. Our data not only provide new insight into the nucleoli in filamentous fungi but also highlight the need for investigating how nucleolar dynamics is regulated in comparison to other eukaryotes.

The emerging role of septins in fungal pathogenesis.

Fungal pathogens undergo specific morphogenetic transitions in order to breach the outer surfaces of plants and invade the underlying host tissue. The ability to change cell shape and switch between non-polarised and polarised growth habits is therefore critical to the lifestyle of plant pathogens. Infection-related development involves remodelling of the cytoskeleton, plasma membrane and cell wall at specific points during fungal pathogenesis. Septin GTPases are components of the cytoskeleton that play pivotal roles in actin remodelling, micron-scale plasma membrane curvature sensing and cell polarity. Septin assemblages, such as rings, collars and gauzes, are known to have important roles in cell shape changes and are implicated in formation of specialised infection structures to enter plant cells. Here, we review and compare the reported functions of septins of plant pathogenic fungi, with a special focus on invasive growth. Finally, we discuss septins as potential targets for broad-spectrum antifungal plant protection strategies.

Hyphopodium-Specific Signaling Is Required for Plant Infection by Verticillium dahliae.

For successful colonization, fungal pathogens have evolved specialized infection structures to overcome the barriers present in host plants. The morphology of infection structures and pathogenic mechanisms are diverse according to host specificity. Verticillium dahliae, a soil-borne phytopathogenic fungus, generates hyphopodium with a penetration peg on cotton roots while developing appressoria, that are typically associated with leaf infection on lettuce and fiber flax roots. In this study, we isolated the pathogenic fungus, V. dahliae (VdaSm), from Verticillium wilt eggplants and generated a GFP-labeled isolate to explore the colonization process of VdaSm on eggplants. We found that the formation of hyphopodium with penetration peg is crucial for the initial colonization of VdaSm on eggplant roots, indicating that the colonization processes on eggplant and cotton share a similar feature. Furthermore, we demonstrated that the VdNoxB/VdPls1-dependent Ca2+ elevation activating VdCrz1 signaling is a common genetic pathway to regulate infection-related development in V. dahliae. Our results indicated that VdNoxB/VdPls1-dependent pathway may be a desirable target to develop effective fungicides, to protect crops from V. dahliae infection by interrupting the formation of specialized infection structures.

Cell Wall Carbohydrate Dynamics during the Differentiation of Infection Structures by the Apple Scab Fungus, Venturia inaequalis.

Scab, caused by the biotrophic fungal pathogen Venturia inaequalis, is the most economically important disease of apples. During infection, V. inaequalis colonizes the subcuticular host environment, where it develops specialized infection structures called runner hyphae and stromata. These structures are thought to be involved in nutrient acquisition and effector (virulence factor) delivery, but also give rise to conidia that further the infection cycle. Despite their importance, very little is known about how these structures are differentiated. Likewise, nothing is known about how these structures are protected from host defenses or recognition by the host immune system. To better understand these processes, we first performed a glycosidic linkage analysis of sporulating tubular hyphae from V. inaequalis developed in culture. This analysis revealed that the V. inaequalis cell wall is mostly composed of glucans (44%) and mannans (37%), whereas chitin represents a much smaller proportion (4%). Next, we used transcriptomics and confocal laser scanning microscopy to provide insights into the cell wall carbohydrate composition of runner hyphae and stromata. These analyses revealed that, during subcuticular host colonization, genes of V. inaequalis putatively associated with the biosynthesis of immunogenic carbohydrates, such as chitin and β-1,6-glucan, are downregulated relative to growth in culture, while on the surface of runner hyphae and stromata, chitin is deacetylated to the less-immunogenic carbohydrate chitosan. These changes are anticipated to enable the subcuticular differentiation of runner hyphae and stromata by V. inaequalis, as well as to protect these structures from host defenses and recognition by the host immune system. IMPORTANCE Plant-pathogenic fungi are a major threat to food security. Among these are subcuticular pathogens, which often cause latent asymptomatic infections, making them difficult to control. A key feature of these pathogens is their ability to differentiate specialized subcuticular infection structures that, to date, remain largely understudied. This is typified by Venturia inaequalis, which causes scab, the most economically important disease of apples. In this study, we show that, during subcuticular host colonization, V. inaequalis downregulates genes associated with the biosynthesis of two immunogenic cell wall carbohydrates, chitin and β-1,6-glucan, and coats its subcuticular infection structures with a less-immunogenic carbohydrate, chitosan. These changes are anticipated to enable host colonization by V. inaequalis and provide a foundation for understanding subcuticular host colonization by other plant-pathogenic fungi. Such an understanding is important, as it may inform the development of novel control strategies against subcuticular plant-pathogenic fungi.

AaCaMKs Positively Regulate Development, Infection Structure Differentiation and Pathogenicity in Alternaria alternata, Causal Agent of Pear Black Spot.

Calcium/calmodulin-dependent protein kinase (CaMK), a key downstream target protein in the Ca2+ signaling pathway of eukaryotes, plays an important regulatory role in the growth, development and pathogenicity of plant fungi. Three AaCaMKs (AaCaMK1, AaCaMK2 and AaCaMK3) with conserved PKC_like superfamily domains, ATP binding sites and ACT sites have been cloned from Alternaria alternata, However, their regulatory mechanism in A. alternata remains unclear. In this study, the function of the AaCaMKs in the development, infection structure differentiation and pathogenicity of A. alternata was elucidated through targeted gene disruption. The single disruption of AaCaMKs had no impact on the vegetative growth and spore morphology but significantly influenced hyphae growth, sporulation, biomass accumulation and melanin biosynthesis. Further expression analysis revealed that the AaCaMKs were up-regulated during the infection structure differentiation of A. alternata on hydrophobic and pear wax substrates. In vitro and in vivo analysis further revealed that the deletion of a single AaCaMKs gene significantly reduced the A. alternata conidial germination, appressorium formation and infection hyphae formation. In addition, pharmacological analysis confirmed that the CaMK specific inhibitor, KN93, inhibited conidial germination and appressorium formation in A. alternata. Meanwhile, the AaCaMKs genes deficiency significantly reduced the A. alternata pathogenicity. These results demonstrate that AaCaMKs regulate the development, infection structure differentiation and pathogenicity of A. alternata and provide potential targets for new effective fungicides.

Melanin synthesis gene Aapks contributes to appressorium formation, stress response, cell well integrity and virulence in Alternaria alternata

Melanin, as a common pigment, performs diverse biological function related to growth and development, environmental stress and pathogenicity in many pathogenic fungi. The polyketide synthase (Aapks) gene is a key gene in 1,8-dihydroxynaphthalene (DHN) melanin synthesis pathway, however, the regulatory roles of Aapks in pear fungal Alternaria alternata, on melanin synthesis, external stresses response, infection structure differentiation and pathogenicity are not clear. The pks gene was cloned and identified in A. alternata. Gene knockout experiment showed that ΔAapks mutant resulted in melanin deficiency and pink pigment production, reduced sporulation and abnormal spore and hyphae. Hypersensitivity to cell wall and oxidant stress factors were also found in ΔAapks, especially the impaired cell wall of ΔAapks was observed after CFW staining, and chitinase activity and related genes expression Aachs4 and Aachs6 were also decreased in ΔAapks. Interestingly, the ΔAapks mutant delayed infection structural differentiation induced by the different physicochemical signals. The virulence of ΔAapks strain was reduced after wound-inoculated pear fruit and non-wound-inoculated tobacco leaves. The above findings suggested the Aapks plays a vital regulatory role in response to stresses, infection structure formation, cell wall integrity and virulence in A. alternata by catalyzing the melanin synthesis.

AaCaM is required for infection structure differentiation and secondary metabolites in pear fungal pathogen Alternaria alternata.

Calmodulin (CaM), acts as a kind of multifunctional Ca2+ sensing protein, which is ubiquitous in fungi, is highly conserved across eukaryotes and is involved in the regulation of a range of physiological processes, including morphogenesis, reproduction and secondary metabolites biosynthesis. Our aim was to understand the characteristics and functions of AaCaM in Alternaria alternata, the causal agent of pear black spot. A 450 bp cDNA sequence of AaCaM gene of A. alternata was cloned by the PCR homology method. Sequence analysis showed that this protein encoded by AaCaM was a stable hydrophilic protein and had a high similarity to Neurospora crassa (CAA50271.1) and other fungi. RT-qPCR analysis determined that AaCaM was differentially upregulated during infection structural differentiation of A. alternata both on hydrophobic and pear wax extract-coated surface, with a 3.37-fold upregulation during the hydrophobic induced appressorium formation period (6h) and a 1.46-fold upregulation during the infection hyphae formation period (8h) following pear wax induction. Pharmaceutical analysis showed that the CaM-specific inhibitor, trifluoperazine (TFP), inhibited spore germination and appressorium formation, and affected toxins and melanin biosynthesis in A. alternata. AaCaM plays an important role in regulating infection structure differentiation and secondary metabolism of A. alternata. Our study provides a theoretical basis for further in-depth investigation of the specific role of AaCaM in the calcium signalling pathway underlying hydrophobic and pear wax-induced infection structure differentiation and pathogenicity of A. alternata.

Molecular Characterization of Phospholipase C in Infection Structure Differentiation Induced by Pear Fruit Surface Signals, Stress Responses, Secondary Metabolism, and Virulence of Alternaria alternata.

Fungal pathogens use plant surface physiochemical signals to trigger specific developmental processes. To assess the role of phospholipase C (PLC) in mediating plant stimuli sensing of Alternaria alternata, the function of three PLC genes was characterized by constructing ΔAaPLC mutants. Here we showed that fruit wax-coated surfaces significantly induced appressorium formation in A. alternata and mutants. Germination of ΔAaPLC mutants did not differ from the wild type. Deletion of AaPLC1 led to the decrease of appressorium formation and infected hyphae, but the degree of reduction varies between the different types of waxes, with the strongest response to pear wax. Appressorium formation and infected hyphae of the ΔAaPLC1 mutant on dewaxed onion epidermis mounted with pear wax (θ4) were reduced by 14.5 and 65.7% after 8 h incubation, while ΔAaPLC2 and ΔAaPLC3 formed the same infection hyphae as wild type. In addition, AaPLC1 mutation caused pleiotropic effects on fungal biological function, including growth deficiency, changes in stress tolerance, weakening of pathogenicity to the host, as well as destruction of mycotoxin synthesis. Both AaPLC2 and AaPLC3 genes were found to have some effects on stress response and mycotoxin production. Taken together, AaPLC genes differentially regulate the growth, stress response, pathogenicity, and secondary metabolism of A. alternata.

The haustorium: The root of biotrophic fungal pathogens.

Biotrophic plant pathogenic fungi are among the dreadful pathogens that continuously threaten the production of economically important crops. The interaction of biotrophic fungal pathogens with their hosts necessitates the development of unique infection mechanisms and involvement of various virulence-associated components. Biotrophic plant pathogenic fungi have an exceptional lifestyle that supports nutrient acquisition from cells of a living host and are fully dependent on the host for successful completion of their life cycle. The haustorium, a specialized infection structure, is the key organ for biotrophic fungal pathogens. The haustorium is not only essential in the uptake of nutrients without killing the host, but also in the secretion and delivery of effectors into the host cells to manipulate host immune system and defense responses and reprogram the metabolic flow of the host. Although there is a number of unanswered questions in this area yet, results from various studies indicate that the haustorium is the root of biotrophic fungal pathogens. This review provides an overview of current knowledge of the haustorium, its structure, composition, and functions, which includes the most recent haustorial transcriptome studies.

The calcineurin-responsive transcription factor Crz1 is required for regulation of infection structure differentiation, calcium homeostasis and cell wall integrity in Alternaria alternata

Ca2+/calcineurin signaling pathway is crucial in regulating growth, stress response and pathogenicity of phytopathogenic fungi. However, there is little information about the downstream calcineurin-responsive zinc finger transcription factor Crz1 in Alternaria alternata, the pathogenic factors of pear black spot. Here, AaCrz1 was identified in A. alternata and its function was characterized by generating target gene knockout strains. The results showed that ΔAaCrz1 was impaired in vegetative growth and spore morphology. The infection structures in the ΔAaCrz1 inhibited on hydrophobic and pear wax extract coated surface. Deletion of AaCrz1 increased sensitivity to different stresses, especially the hypersensitivity to Ca2+. In addition, AaPMC1 and AaPMR1 genes were regulated by AaCrz1 in response to Ca2+ level. CFW staining indicated that the cell wall of ΔAaCrz1 was damaged, and the ΔAaCrz1 displayed a reduction in the expression of genes related to cell wall biosynthesis, such as chitin synthases (CHS) and β-1,3-glucan synthase (FKS). Furthermore, AaCrz1 was involved in the full virulence of A. alternata on pear fruit. Collectively, this study suggests that identified AaCrz1 is essential for the development, infection structure formation, calcium homeostasis, cell wall integrity and virulence of A. alternata.

Transcriptional regulation of secondary metabolism and autophagy genes in response to DNA replication stress in Setosphaeria turcica

The fungal pathogen Setosphaeria turcica causes northern corn leaf blight (NCLB), which leads to considerable crop losses. Setosphaeria turcica elaborates a specialized infection structures called appressorium for maize infection. Previously, we demonstrated that the S. turcica triggers an S-phase checkpoint and ATR (Ataxia Telangiectasia and Rad3 related)-dependent self-protective response to DNA genotoxic insults during maize infection. However, how the regulatory mechanism works was still largely unknown. Here, we report a genome wide transcriptional profile analysis during appressorium formation in the present of DNA replication stress. We performed RNA-Seq analysis to identify S. tuicica genes responsive to DNA replication stress. In the current work, we found that appressorium-mediated maize infection by S. turcica is significantly blocked by S-phase checkpoint. A large serial of secondary metabolite and melanin biosynthesis genes were blocked in appressorium formation of S. turcica during the replication stress. The secondary metabolite biosynthesis genes including alcohol dehydrogenase GroES-like domain, multicopper oxidase, ABC-transporter families, cytochrome P450 and FAD-containing monooxygenase were related to plant pathogen infection. In addition, we demonstrated that autophagy in S. turcica is up-regulated by ATR as a defense response to stress. We identified StATG3, StATG4, StATG5, StATG7 and StATG16 genes for autophagy were induced by ATR-mediated S-phase checkpoint. We therefore propose that in response to genotoxic stress, S. turcica utilizes ATR-dependent pathway to turn off transcription of genes governing appressorium-mediated infection, and meanwhile inducing transcription of autophagy genes likely as a mechanism of self-protection, aside from the more conservative responses in eukaryotes.

The MoPah1 phosphatidate phosphatase is involved in lipid metabolism, development, and pathogenesis in Magnaporthe oryzae.

As with the majority of the hemibiotrophic fungal pathogens, the rice blast fungus Magnaporthe oryzae uses highly specialized infection structures called appressoria for plant penetration. Appressoria differentiated from germ tubes rely on enormous turgor pressure to directly penetrate the plant cell, in which process lipid metabolism plays a critical role. In this study, we characterized the MoPAH1 gene in M. oryzae, encoding a putative highly conserved phosphatidate phosphatase. The expression of MoPAH1 was up‐regulated during plant infection. The MoPah1 protein is expressed at all developmental and infection stages, and is localized to the cytoplasm. Disruption of MoPAH1 causes pleiotropic defects in vegetative growth, sporulation, and heat tolerance. The lipid profile is significantly altered in the Mopah1 mutant. Lipidomics assays showed that the level of phosphatidic acid (PA) was increased in the mutant, which had reduced levels of diacylglycerol and triacylglycerol. Using a PA biosensor, we showed that the increased level of PA in the Mopah1 mutant was primarily accumulated in the vacuole. The Mopah1 mutant was blocked in both conidiation and the formation of appressorium‐like structures at hyphal tips. It was nonpathogenic and failed to cause any blast lesions on rice and barley seedlings. RNA sequencing analysis revealed that MoPah1 regulates the expression of transcription factors critical for various developmental and infection‐related processes. The Mopah1 mutant was reduced in the expression and phosphorylation of Pmk1 MAP kinase and delayed in autophagy. Our study demonstrates that MoPah1 is necessary for lipid metabolism, fungal development, and pathogenicity in M. oryzae.

Melanin, DNA replication, and autophagy affect appressorium development in Setosphaeria turcica by regulating glycerol accumulation and metabolism

Setosphaeria turcica (syn. Exserohilum turcicum) is the pathogenic fungus of maize (Zea mays) that causes northern leaf blight, which is a major maize disease worldwide. Melanized appressoria are highly specialized infection structures formed by germinated conidia of S. turcica that infect maize leaves. The appressorium penetrates the plant cuticle by generating turgor, and glycerol is known to be the main source of the turgor. Here, the infection position penetrated by the appressorium on maize leaves was investigated, most of the germinated conidia entered the leaf interior by directly penetrating the epidermal cells, and the appressorium structure was necessary for the infection, whether it occurred through epidermal cells or stomata. Then, to investigate the effects of key factors in the development of the appressorium, we studied the effects of three inhibitors, including a melanin inhibitor (tricyclazole, TCZ), a DNA replication inhibitor (hydroxyurea, HU), and an autophagy inhibitor (3-methyladenine, 3-MA), on appressorium turgor and glycerol content. As results, appressorium turgor pressure and glycerol concentration in the appressorium reached their highest levels at the mature stage of the appressorium under the control and inhibitor treatments. The three inhibitors had the greatest effects on appressorium turgor pressure at this stage. Glycogen and liposomes are the main substances producing glycerol. It was also found inhibitors affected the distribution of glycogen and liposomes, which were detected in the conidia, the germ tube, and the appressorium during appressorium development. This study provides profound insight into the relationship between appressorium turgor pressure and glycerol content, which was affected by the synthesis of melanin, DNA replication, and autophagy in the developing appressorium during a S. turcica infection.

Magnaporthe oryzae Transcription Factor MoBZIP3 Regulates Appressorium Turgor Pressure Formation during Pathogenesis

The devastating fungus Magnaporthe oryzae (M. oryzae) forms a specialized infection structure known as appressorium, which generates enormous turgor, to penetrate the plant cells. However, how M. oryzae regulates the appressorium turgor formation, is not well understood. In this study, we identified MoBZIP3, a bZIP transcription factor that functioned in pathogenesis in M. oryzae. We found that the pathogenicity of the MoBZIP3 knockout strain (Δmobzip3) was significantly reduced, and the defect was restored after re-expression of MoBZIP3, indicating that MoBZIP3 is required for M. oryzae virulence. Further analysis showed that MoBZIP3 functions in utilization of glycogen and lipid droplets for generation of glycerol in appressorium. MoBZIP3 localized in the nucleus and could bind directly to the promoters of the glycerol synthesis-related genes, MoPTH2, MoTGL1 and MoPEX6, and regulate their expression which is critical for glycerol synthesis in the appressorium turgor pressure generation. Furthermore, the critical turgor sensor gene MoSln1 was also down regulated and its subcellular localization was aberrant in Δmobzip3, which leads to a disordered actin assembly in the Δmobzip3 appressorium. Taken together, these results revealed new regulatory functions of the bZIP transcription factor MoBZIP3, in regulating M. oryzae appressorium turgor formation and infection.

The transmembrane protein AaSho1 is essential for appressorium formation and secondary metabolism but dispensable for vegetative growth in pear fungal Alternaria alternata

The high-osmolarity glycerol response (HOG) pathway is pivotal in environmental stress response, differentiation and virulence of Alternaria alternata. The synthetic high osmolarity sensitive sensor Sho1 has been postulated to regulate the HOG pathway. To determine the regulatory role of transmembrane protein Sho1 on vegetative growth, secondary metabolism and infection structure formation, a gene (AaSho1) encoding Sho1 was cloned and characterized from A. alternata (JT-03). Sequence analysis showed that AaSho1 has all four characteristic transmembrane domains and the SH3 domain present in another Sho1 gene from several filamentous fungal. The quantitative RT-PCR analysis showed that fruit wax extract significantly up-regulated AaSho1 gene expression in vitro. Pharmacological experiments showed that A. alternata treated with nystatin, a specific AaSho1 inhibitor, had no significant effect on the morphology of A. alternata and the invasive growth in pear fruit. However, nystatin treatment significantly reduced spore germination rates on different wax-coated hydrophobic surfaces, with 58.00, 46.70 and 83.72% reduced for fruit wax, beeswax and paraffin coated. Meanwhile, the secondary metabolism altenuene (ALT), tentoxin (TEN) toxin, and melanin content were also affected by nystatin treatment. These findings suggest that AaSho1 is required for the infection structure differentiation and secondary metabolism of A. alternata in response to physiochemical signals on the host surfaces.