Csn5 inhibits autophagy by regulating the ubiquitination of Atg6 and Tor to mediate the pathogenicity of Magnaporthe oryzae.

Csn5 is subunit 5 of the COP9 signalosome (CSN), but the mechanism by which it strictly controls the pathogenicity of pathogenic fungi through autophagy remains unclear. Here, we found that Csn5 deficiency attenuated pathogenicity and enhanced autophagy in Magnaporthe oryzae. MoCSN5 knockout led to overubiquitination and overdegradation of MoTor (the core protein of the TORC1 complex [target of rapamycin]) thereby promoted autophagy. In addition, we identified MoCsn5 as a new interactor of MoAtg6. Atg6 was found to be ubiquitinated through linkage with lysine 48 (K48) in cells, which is necessary for infection-associated autophagy in pathogenic fungi. K48-ubiquitination of Atg6 enhanced its degradation and thereby inhibited autophagic activity. Our experimental results indicated that MoCsn5 promoted K48-ubiquitination of MoAtg6, which reduced the MoAtg6 protein content and thus inhibited autophagy. Aberrant ubiquitination and autophagy in ΔMocsn5 led to pleiotropic defects in the growth, development, stress resistance, and pathogenicity of M. oryzae. In summary, our study revealed a novel mechanism by which Csn5 regulates autophagy and pathogenicity in rice blast fungus through ubiquitination.

The MoLfa1 Protein Regulates Fungal Development and Septin Ring Formation in Magnaporthe oryzae.

Septins play a key regulatory role in cell division, cytokinesis, and cell polar growth of the rice blast fungus (Magnaporthe oryzae). We found that the organization of the septin ring, which is essential for appressorium-mediated infection in M. oryzae, requires long-chain fatty acids (LCFAs), which act as mediators of septin organization at membrane interfaces. However, it is unclear how septin ring formation and LCFAs regulate the pathogenicity of the rice blast fungus. In this study, a novel protein was named MoLfa1 because of its role in LCFAs utilization. MoLfa1 affects the utilization of LCFAs, lipid metabolism, and the formation of the septin ring by binding with phosphatidylinositol phosphates (PIPs), thereby participating in the construction of penetration pegs of M. oryzae. In addition, MoLfa1 is localized in the endoplasmic reticulum (ER) and interacts with the ER-related protein MoMip11 to affect the phosphorylation level of Mps1. (Mps1 is the core protein in the MPS1-MAPK pathway.) In conclusion, MoLfa1 affects conidia morphology, appressorium formation, lipid metabolism, LCFAs utilization, septin ring formation, and the Mps1-MAPK pathway of M. oryzae, influencing pathogenicity.

Roles of CcDFR and CcOMT9 in the cyanidin biosynthesis and development of Cordyceps cicadae.

Introduction: Cordyceps cicadae is a traditional Chinese medicinal fungus known for its rich production of bioactive substances, particularly cyanidin, an anthocyanin commonly found in plants with notable anti-inflammatory, anti-tumor, antiviral, and antibacterial properties. This study revealed two key genes, CcDFR and CcOMT9, affecting cyanidin biosynthesis in C. cicadae. Methods: The roles of these genes in cyanidin production, growth, and development were elucidated through the gene knockout method, phenotypic analysis, transcriptomics, and metabolomics. Results: CcDFR deletion led to reduced cyanidin-3-O-glucoside (C3G), suppressed expression of cyanidin biosynthesis genes, impaired synnemata formation, decreased polysaccharide and adenosine content, and diminished chitinase activity. Meanwhile, the ΔCcOMT9 mutant exhibited an increase in C3G production, promoted expression of cyanidin biosynthesis genes and rising bioactive compounds, suppressed RNA methylation, and led to phenylalanine accumulation with no effect on fruiting body formation. Discussion: We revealed a distinct anthocyanin biosynthesis pathway in C. cicadae and identified two genes with opposite functions, laying the foundation for future genetic modification of cyanidin-producing strains using modern biological techniques. This will shorten the production period of this valuable compound, facilitating the industrial-scale production of cyanidin.

The cell cycle, autophagy, and cell wall integrity pathway jointly governed by MoSwe1 in Magnaporthe oryzae.

The cell cycle is pivotal to cellular differentiation in plant pathogenic fungi. Cell wall integrity (CWI) signaling plays an essential role in coping with cell wall stress. Autophagy is a degradation process in which cells decompose their components to recover macromolecules and provide energy under stress conditions. However, the specific association between cell cycle, autophagy and CWI pathway remains unclear in model pathogenic fungi Magnaporthe oryzae. Here, we have identified MoSwe1 as the conserved component of the cell cycle in the rice blast fungus. We have found that MoSwe1 targets MoMps1, a conserved critical MAP kinase of the CWI pathway, through protein phosphorylation that positively regulates CWI signaling. The CWI pathway is abnormal in the ΔMoswe1 mutant with cell cycle arrest. In addition, we provided evidence that MoSwe1 positively regulates autophagy by interacting with MoAtg17 and MoAtg18, the core autophagy proteins. Moreover, the S phase initiation was earlier, the morphology of conidia and appressoria was abnormal, and septum formation and glycogen degradation were impaired in the ΔMoswe1 mutant. Our research defines that MoSWE1 regulation of G1/S transition, CWI pathway, and autophagy supports its specific requirement for appressorium development and virulence in plant pathogenic fungi. Video Abstract.

SP-141 targets Trs85 to inhibit rice blast fungus infection and functions as a potential broad-spectrum antifungal agent.

Rice blast is a devastating disease worldwide, threatening rice production and food security. The blast fungus Magnaporthe oryzae invades the host via the appressorium, a specialized pressure-generating structure that generates enormous turgor pressure to penetrate the host cuticle. However, owing to ongoing evolution of fungicide resistance, it is vitally important to identify new targets and fungicides. Here, we show that Trs85, a subunit of the transport protein particle III complex, is essential for appressorium-mediated infection in M. oryzae. We explain how Trs85 regulates autophagy through Ypt1 (a small guanosine triphosphatase protein) in M. oryzae. We then identify a key conserved amphipathic α helix within Trs85 that is associated with pathogenicity of M. oryzae. Through computer-aided screening, we identify a lead compound, SP-141, that affects autophagy and the Trs85-Ypt1 interaction. SP-141 demonstrates a substantial capacity to effectively inhibit infection caused by the rice blast fungus while also exhibiting wide-ranging potential as an antifungal agent with broad-spectrum activity. Taken together, our data show that Trs85 is a potential new target and that SP-141 has potential for the control of rice blast. Our findings thus provide a novel strategy that may help in the fight against rice blast.

Recent Advances in Effector Research of Magnaporthe oryzae.

Recalcitrant rice blast disease is caused by Magnaporthe oryzae, which has a significant negative economic reverberation on crop productivity. In order to induce the disease onto the host, M. oryzae positively generates many types of small secreted proteins, here named as effectors, to manipulate the host cell for the purpose of stimulating pathogenic infection. In M. oryzae, by engaging with specific receptors on the cell surface, effectors activate signaling channels which control an array of cellular activities, such as proliferation, differentiation and apoptosis. The most recent research on effector identification, classification, function, secretion, and control mechanism has been compiled in this review. In addition, the article also discusses directions and challenges for future research into an effector in M. oryzae.

The biological functions of sphingolipids in plant pathogenic fungi.

Sphingolipids are critically significant in a range of biological processes in animals, plants, and fungi. In mammalian cells, they serve as vital components of the plasma membrane (PM) in maintaining its structure, tension, and fluidity. They also play a key role in a wide variety of biological processes, such as intracellular signal transduction, cell polarization, differentiation, and migration. In plants, sphingolipids are important for cell development and for cell response to environmental stresses. In pathogenic fungi, sphingolipids are crucial for the initiation and the development of infection processes afflicting humans. However, our knowledge on the metabolism and function of the sphingolipid metabolic pathway of pathogenic fungi affecting plants is still very limited. In this review, we discuss recent developments on sphingolipid pathways of plant pathogenic fungi, highlighting their uniqueness and similarity with plants and animals. In addition, we discuss recent advances in the research and development of fungal-targeted inhibitors of the sphingolipid pathway, to gain insights on how we can better control the infection process occurring in plants to prevent or/and to treat fungal infections in crops.

Transcriptomic and Metabolomic Investigation on Leaf Necrosis Induced by ZmWus2 Transient Overexpression in Nicotiana benthamiana.

WUSCHEL (WUS) is a crucial transcription factor in regulating plant stem cell development, and its expression can also improve genetic transformation. However, the ectopic expression of WUS always causes pleiotropic effects during genetic transformation, making it important to understand the regulatory mechanisms underlying these phenomena. In our study, we found that the transient expression of the maize WUS ortholog ZmWus2 caused severe leaf necrosis in Nicotiana benthamiana. We performed transcriptomic and non-target metabolomic analyses on tobacco leaves during healthy to wilted states after ZmWus2 transient overexpression. Transcriptomic analysis revealed that ZmWus2 transformation caused active metabolism of inositol trisphosphate and glycerol-3-phosphate, while also upregulating plant hormone signaling and downregulating photosystem and protein folding pathways. Metabolomic analysis mainly identified changes in the synthesis of phenylpropanoid compounds and various lipid classes, including steroid synthesis. In addition, transcription factors such as ethylene-responsive factors (ERFs), the basic helix-loop-helix (bHLH) factors, and MYBs were found to be regulated by ZmWus2. By integrating these findings, we developed a WUS regulatory model that includes plant hormone accumulation, stress responses, lipid remodeling, and leaf necrosis. Our study sheds light on the mechanisms underlying WUS ectopic expression causing leaf necrosis and may inform the development of future genetic transformation strategies.

The triglyceride catabolism regulated by a serine/threonine protein phosphatase, Smek1, is required for development and plant infection in Magnaporthe oryzae.

Magnaporthe oryzae is a pathogenic fungus that seriously harms rice production. Phosphatases and carbon metabolism play crucial roles in the growth and development of eukaryotes. However, it remains unclear how serine/threonine phosphatases regulate the catabolism of triglycerides, a major form of stored lipids. In this study, we identified a serine/threonine protein phosphatase regulatory subunit, Smek1, which is required for the growth, conidiation, and virulence of M. oryzae. Deletion of SMEK1 led to defects in the utilization of lipids, arabinose, glycerol, and ethanol. In glucose medium, the expression of genes involved in lipolysis, long-chain fatty acid degradation, ß-oxidation, and the glyoxylate cycle increased in the Δsmek1 mutant, which is consistent with ΔcreA in which a carbon catabolite repressor CREA was deleted. In lipid medium, the expression of genes involved in long-chain fatty acid degradation, ß-oxidation, the glyoxylate cycle, and utilization of arabinose, ethanol, or glycerol decreased in the Δsmek1 mutant, which is consistent with Δcrf1 in which a transcription activator CRF1 required for carbon metabolism was deleted. Lipase activity, however, increased in the Δsmek1 mutant in both glucose and lipid media. Moreover, Smek1 directly interacted with CreA and Crf1, and dephosphorylated CreA and Crf1 in vivo. The phosphatase Smek1 is therefore a dual-function regulator of the lipid and carbohydrate metabolism, and controls fungal development and virulence by coordinating the functions of CreA and Crf1 in carbon catabolite repression (CCR) and derepression (CCDR).

MoCbp7, a Novel Calcineurin B Subunit-Binding Protein, Is Involved in the Calcium Signaling Pathway and Regulates Fungal Development, Virulence, and ER Homeostasis in Magnaporthe oryzae.

Calcineurin, a key regulator of the calcium signaling pathway, is involved in calcium signal transduction and calcium ion homeostasis. Magnaporthe oryzae is a devastating filamentous phytopathogenic fungus in rice, yet little is known about the function of the calcium signaling system. Here, we identified a novel calcineurin regulatory-subunit-binding protein, MoCbp7, which is highly conserved in filamentous fungi and was found to localize in the cytoplasm. Phenotypic analysis of the MoCBP7 gene deletion mutant (ΔMocbp7) showed that MoCbp7 influenced the growth, conidiation, appressorium formation, invasive growth, and virulence of M. oryzae. Some calcium-signaling-related genes, such as YVC1, VCX1, and RCN1, are expressed in a calcineurin/MoCbp7-dependent manner. Furthermore, MoCbp7 synergizes with calcineurin to regulate endoplasmic reticulum homeostasis. Our research indicated that M. oryzae may have evolved a new calcium signaling regulatory network to adapt to its environment compared to the fungal model organism Saccharomyces cerevisiae.

Acyl-coenzyme A binding protein MoAcb1 regulates conidiation and pathogenicity in Magnaporthe oryzae.

Magnaporthe oryzae is a filamentous fungus that causes rice blast. Rice blast seriously threatens the safety of food production. The normal synthesis and metabolism of fatty acids are extremely important for eukaryotes, and acyl-CoA is involved in fatty acid metabolism. Acyl-CoA binding (ACB) proteins specifically bind both medium-chain and long-chain acyl-CoA esters. However, the role of the Acb protein in plant-pathogenic fungi has not yet been investigated. Here, we identified MoAcb1, a homolog of the Acb protein in Saccharomyces cerevisiae. Disruption of MoACB1 causes delayed hyphal growth, significant reduction in conidial production and delayed appressorium development, glycogen availability, and reduced pathogenicity. Using immunoblotting and chemical drug sensitivity analysis, MoAcb1 was found to be involved in endoplasmic reticulum autophagy (ER-phagy). In conclusion, our results suggested that MoAcb1 is involved in conidia germination, appressorium development, pathogenicity and autophagy processes in M. oryzae.

Model construction for height to crown base of Larix olgensis based on mixed-effects model and quantile regression.

Height to crown base is an important index reflecting the characteristics of tree crown. It is of great significance to accurately quantify height to crown base for forest management and increasing stand production. We used nonlinear regression to construct the height to crown base generalized basic model, and further extended that to the mixed-effects model and quantile regression model. The prediction ability of the models was evaluated and compared by the 'leave-one-out' cross-validate. Four sampling designs and different sampling sizes were used to calibrate the height to crown base model, and the best model calibration scheme was selected. The results showed that based on the height to crown base generalized model including tree height, diameter at breast height, basal area of the stand and average dominant height, the prediction accuracy of the expanded mixed-effects model and the combined three-quartile regression model were obviously improved. The mixed-effects model was slightly better than the combined three-quartile regression model, and the optimal sampling calibration scheme was to select five average trees. The mixed-effects model with five average trees was recommended to predict the height to crown base in practice.

MoVast2 combined with MoVast1 regulates lipid homeostasis and autophagy in Magnaporthe oryzae.

Macroautophagy/autophagy is an evolutionarily conserved biological process among eukaryotes that degrades unwanted materials such as protein aggregates, damaged mitochondria and even viruses to maintain cell survival. Our previous studies have demonstrated that MoVast1 acts as an autophagy regulator regulating autophagy, membrane tension, and sterol homeostasis in rice blast fungus. However, the detailed regulatory relationships between autophagy and VASt domain proteins remain unsolved. Here, we identified another VASt domain-containing protein, MoVast2, and further uncovered the regulatory mechanism of MoVast2 in M. oryzae. MoVast2 interacted with MoVast1 and MoAtg8, and colocalized at the PAS and deletion of MoVAST2 results in inappropriate autophagy progress. Through TOR activity analysis, sterols and sphingolipid content detection, we found high sterol accumulation in the ΔMovast2 mutant, whereas this mutant showed low sphingolipids and low activity of both TORC1 and TORC2. In addition, MoVast2 colocalized with MoVast1. The localization of MoVast2 in the MoVAST1 deletion mutant was normal; however, deletion of MoVAST2 leads to mislocalization of MoVast1. Notably, the wide-target lipidomic analyses revealed significant changes in sterols and sphingolipids, the major PM components, in the ΔMovast2 mutant, which was involved in lipid metabolism and autophagic pathways. These findings confirmed that the functions of MoVast1 were regulated by MoVast2, revealing that MoVast2 combined with MoVast1 maintained lipid homeostasis and autophagy balance by regulating TOR activity in M. oryzae.

Current opinions on mitophagy in fungi.

Mitophagy, as one of the most important cellular processes to ensure quality control of mitochondria, aims at transporting damaged, aging, dysfunctional or excess mitochondria to vacuoles (plants and fungi) or lysosomes (mammals) for degradation and recycling. The normal functioning of mitophagy is critical for cellular homeostasis from yeasts to humans. Although the role of mitophagy has been well studied in mammalian cells and in certain model organisms, especially the budding yeast Saccharomyces cerevisiae, our understanding of its significance in other fungi, particularly in pathogenic filamentous fungi, is still at the preliminary stage. Recent studies have shown that mitophagy plays a vital role in spore production, vegetative growth and virulence of pathogenic fungi, which are very different from its roles in mammal and yeast. In this review, we summarize the functions of mitophagy for mitochondrial quality and quantity control, fungal growth and pathogenesis that have been reported in the field of molecular biology over the past two decades. These findings may help researchers and readers to better understand the multiple functions of mitophagy and provide new perspectives for the study of mitophagy in fungal pathogenesis.Abbreviations: AIM/LIR: Atg8-family interacting motif/LC3-interacting region; BAR: Bin-Amphiphysin-Rvs; BNIP3: BCL2 interacting protein 3; CK2: casein kinase 2; Cvt: cytoplasm-to-vacuole targeting; ER: endoplasmic reticulum; IMM: inner mitochondrial membrane; mETC: mitochondrial electron transport chain; OMM: outer mitochondrial membrane; OPTN: optineurin; PAS: phagophore assembly site; PD: Parkinson disease; PE: phosphatidylethanolamine; PHB2: prohibitin 2; PX: Phox homology; ROS, reactive oxygen species; TM: transmembrane.

GPI-Anchored Protein Homolog IcFBR1 Functions Directly in Morphological Development of Isaria cicadae.

Isaria cicadae is a famous edible and medicinal fungus in China and Asia. The molecular basis of morphogenesis and synnemal formation needs to be understood in more detail because this is the main source of biomass production in I. cicadae. In the present study, a fruiting body formation-related gene with a glycosylphosphatidylinositol (GPI) anchoring protein (GPI-Ap) gene homolog IcFBR1 was identified by screening random insertion mutants. Targeted deletion of IcFBR1 resulted in abnormal formation of synnemata, impairing aerial hyphae growth and sporulation. The IcFBR1 mutants were defective in the utilization of carbon sources with reduced polysaccharide contents and the regulation of amylase and protease activities. Transcriptome analysis of ΔIcfbr1 showed that IcFBR1 deletion influenced 49 gene ontology terms, including 23 biological processes, 9 molecular functions, and 14 cellular components. IcFBR1 is therefore necessary for regulating synnemal development, secondary metabolism, and nutrient utilization in this important edible and medicinal fungus. This is the first report illustrating that the function of IcFBR1 is associated with the synnemata in I. cicadae.

A Subunit of the COP9 Signalosome, MoCsn6, Is Involved in Fungal Development, Pathogenicity, and Autophagy in Rice Blast Fungus.

The COP9 signalosome (CSN) is a highly conserved protein complex in eukaryotes, affecting various development and signaling processes. To date, the biological functions of the COP9 signalosome and its subunits have not been determined in Magnaporthe oryzae. In this study, we characterized the CSN in M. oryzae (which we named MoCsn6) and analyzed its biological functions. MoCsn6 is involved in fungal development, autophagy, and plant pathogenicity. Compared with the wild-type strain 70-15, ΔMocsn6 mutants showed a significantly reduced growth rate, sporulation rate, and germ tube germination rate. Pathogenicity assays showed that the ΔMocsn6 mutants did not cause or significantly reduced the number of disease spots on isolated barley leaves. After the MoCSN6 gene was complemented into the ΔMocsn6 mutant, vegetative growth, sporulation, and pathogenicity were restored. The Osm1 and Pmk1 phosphorylation pathways were also disrupted in the ΔMocsn6 mutants. Furthermore, we found that MoCsn6 participates in the autophagy pathway by interacting with the autophagy core protein MoAtg6 and regulating its ubiquitination level. Deletion of MoCSN6 resulted in rapid lipidation of MoAtg8 and degradation of the autophagic marker protein green fluorescent protein-tagged MoAtg8 under nutrient and starvation conditions, suggesting that MoCsn6 negatively regulates autophagic activity. Taken together, our results demonstrate that MoCsn6 plays a crucial role in regulating fungal development, pathogenicity, and autophagy in M. oryzae. IMPORTANCE Magnaporthe oryzae, a filamentous fungus, is the cause of many cereal diseases. Autophagy is involved in fungal development and pathogenicity. The COP9 signalosome (CSN) has been extensively studied in ubiquitin pathways, but its regulation of autophagy has rarely been reported in plant-pathogenic fungi. Investigations on the relationship between CSN and autophagy will deepen our understanding of the pathogenic mechanism of M. oryzae and provide new insights into the development of new drug targets to control fungal diseases. In this study, the important function of Csn6 in the autophagy regulation pathway and its impact on the pathogenicity of M. oryzae were determined. We showed that Csn6 manages autophagy by interacting with the autophagy core protein Atg6 and regulating its ubiquitination level. Furthermore, future investigations that explore the function of CSN will deepen our understanding of autophagy mechanisms in rice blast fungus.

The crucial role of the regulatory mechanism of the Atg1/ULK1 complex in fungi.

Autophagy, an evolutionarily conserved cellular degradation pathway in eukaryotes, is hierarchically regulated by autophagy-related genes (Atgs). The Atg1/ULK1 complex is the most upstream factor involved in autophagy initiation. Here，we summarize the recent studies on the structure and molecular mechanism of the Atg1/ULK1 complex in autophagy initiation, with a special focus on upstream regulation and downstream effectors of Atg1/ULK1. The roles of pathogenicity and autophagy aspects in Atg1/ULK1 complexes of various pathogenic hosts, including plants, insects, and humans, are also discussed in this work based on recent research findings. We establish a framework to study how the Atg1/ULK1 complex integrates the signals that induce autophagy in accordance with fungus to mammalian autophagy regulation pathways. This framework lays the foundation for studying the deeper molecular mechanisms of the Atg1 complex in pathogenic fungi.

Genome-Wide Analysis of AGC Kinases Reveals that MoFpk1 Is Required for Development, Lipid Metabolism, and Autophagy in Hyperosmotic Stress of the Rice Blast Fungus Magnaporthe oryzae.

During eukaryotic evolution, the TOR-AGC kinase signaling module is involved in the coordinated regulation of cell growth and survival. However, the AGC kinases in plant-pathogenic fungi remain poorly understood. In this study, we have identified 20 members of the AGC family of protein kinases. Evolutionary and biological studies have revealed that AGC kinases are highly conserved and involved in the growth (8 genes), conidiation (13 genes), conidial germination (9 genes), appressorium formation (9 genes), and pathogenicity (5 genes) of Magnaporthe oryzae, in which a subfamily protein of the AGC kinases, MoFpk1, the activator of flippase, specifically exhibited diverse roles. Two kinase sites were screened and found to be critical for MoFpk1: 230K and 326D. Moreover, MoFpk1 is involved in cell wall integrity through the negative regulation of MoMps1 phosphorylation. The deletion of MoFpk1 resulted in defective phosphatidylacetamide (PE) and phosphatidylserine (PS) turnover and a series of lipid metabolism disorders. Under hyperosmotic stress, since the ΔMofpk1 mutant is unable to maintain membrane asymmetry, MoYpk1 phosphorylation and MoTor activity were downregulated, thus enhancing autophagy. Our results provide insights into the evolutionary and biological relationships of AGC kinases and new insight into plasma membrane (PM) homeostasis, i.e., responses to membrane stress and autophagy through lipid asymmetry maintenance. IMPORTANCE Our identification and analysis of evolutionary and biological relationships provide us with an unprecedented high-resolution view of the flexible and conserved roles of the AGC family in the topmost fungal pathogens that infect rice, wheat, barley, and millet. Guided by these insights, an AGC member, MoFpk1, was found to be indispensable for M. oryzae development. Our study defined a novel mechanism of plasma membrane homeostasis, i.e., adaptation to stress through the asymmetric distribution of phospholipids. Furthermore, defects in the asymmetric distribution of phospholipids in the membrane enhanced autophagy under hyperosmotic stress. This study provides a new mechanism for the internal linkage between lipid metabolism and autophagy, which may help new fungicide target development for controlling this devastating disease.

An appressorium membrane protein, Pams1, controls infection structure maturation and virulence via maintaining endosomal stability in the rice blast fungus.

The rice blast fungus Magnaporthe oryzae spores differentiate and mature into functional appressoria by sensing the host surface signals. Environmental stimuli are transduced into cells through internalization during appressorium formation, such as in the cAMP-PKA pathway. Here, we describe a novel contribution to how appressoria mature on the surface of a leaf, and its connection to endosomes and the cAMP-PKA pathway. An appressorium membrane-specific protein, Pams1, is required for maintaining endosomal structure, appressorium maturation, and virulence in M. oryzae. During appressorium development, Pams1 was translocated from the cell membrane to the endosomal membrane. Deletion of PAMS1 led to the formation of two types of abnormal appressoria after 8 h post inoculation (hpi): melanized type I had a reduced virulence, while pale type II was dead. Before 8 hpi, Δpams1 formed appressoria that were similar to those of the wild type. After 8 hpi, the appressoria of Δpams1 was differentiated into two types: (1) the cell walls of type I appressoria were melanized, endosomes were larger, and had a different distribution from the wild type and (2) Type II appressoria gradually stopped melanization and began to die. The organelles, including the nucleus, endosomes, mitochondria, and endoplasmic reticula, were degraded, leaving only autophagic body-like vesicles in type II appressoria. The addition of exogenous cAMP to Δpams1 led to the formation of a greater proportion of type I appressoria and a smaller proportion of type II appressoria. Thus, defects in endosomal structure and the cAMP-PKA pathway are among the causes of the defective appressorium maturation and virulence of Δpams1.

The peroxins BcPex8, BcPex10, and BcPex12 are required for the development and pathogenicity of Botrytis cinerea.

Peroxisomes have been proved playing roles in infection of several plant pathogens. Although the contribution of a portion of peroxins in pathogenicity was demonstrated, most of them are undocumented in fungi, especially, Botrytis cinerea. The homologs of Pex8, Pex10, and Pex12 in B. cinerea were functionally characterized in this work using gene disruption strategies. Compared with the wild-type strain (WT), the Δbcpex8, Δbcpex10, and Δbcpex12 mutants exhibited significant reduction in melanin production, fatty acid utilization, and decreased tolerance to high osmotic pressure and reactive oxygen species (ROS). The mycelial growth and conidiation of were significantly inhibited in Δbcpex8, Δbcpex10, and Δbcpex12 strains. The mycelial growth rates of Δbcpex8, Δbcpex10, and Δbcpex12 were reduced by 32, 35, and 34%, respectively, compared with WT and ectopic transformant (ET), and the conidiation was reduced by approximately 89, 27, and 88%, respectively. The conidial germination, germ tube elongation, and the formation of initiate infection structures (IFSs) were also reduced by the deletion of the genes. The pathogenicity was tested on the leaves of tobacco and strawberry, and fruits of tomato. On the leaves of tobacco and strawberry, the Δbcpex8, Δbcpex10, and Δbcpex12 mutants could not induce necrotic lesions, and the lesions on tomato fruits infected with the mutants were significantly reduced than those of the wide type. The results indicated that BcPEX8, BcPEX10, and BcPEX12 are indispensable for the development and pathogenicity of B. cinerea.