Comparative Mitogenomics Analysis Revealed Evolutionary Divergence among Neopestalotiopsis Species Complex (Fungi: Xylariales).

The genus Neopestalotiopsis consists of obligate parasites that cause ring spot, scab, and leaf blight diseases in higher plant species. We assembled the three complete mitogenomes for the guava fruit ring spot pathogen, Neopestalotiopsis cubana. The mitogenomes are circular, with sizes of 38,666 bp, 33,846 bp, and 32,593 bp. The comparative analyses with Pestalotiopsis fici showed that N. cubana differs greatly from it in the length of the mitogenomes and the number of introns. Moreover, they showed significant differences in the gene content and tRNAs. The two genera showed little difference in gene skewness and codon preference for core protein-coding genes (PCGs). We compared gene sequencing in the mitogenomes of the order Xylariales and found large-scale gene rearrangement events, such as gene translocations and the duplication of tRNAs. N. cubana shows a unique evolutionary position in the phylum Ascomycota constructed in phylogenetic analyses. We also found a more concentrated distribution of evolutionary pressures on the PCGs of Neopestalotiopsis in the phylum Ascomycota and that they are under little selective pressure compared to other species and are subjected to purifying selection. This study explores the evolutionary dynamics of the mitogenomes of Neopestalotiopsis and provides important support for genetic and taxonomic studies.

Bayogenin 3-O-cellobioside confers non-cultivar-specific defence against the rice blast fungus Pyricularia oryzae.

Rice cultivars from japonica and indica lineage possess differential resistance against blast fungus as a result of genetic divergence. Whether different rice cultivars also show distinct metabolomic changes in response to P. oryzae, and their role in host resistance, are poorly understood. Here, we examine the responses of six different rice cultivars from japonica and indica lineage challenged with P. oryzae. Both susceptible and resistant rice cultivars expressed several metabolites exclusively during P. oryzae infection, including the saponin Bayogenin 3-O-cellobioside. Bayogenin 3-O-cellobioside level in infected rice directly correlated with their resistant attributes. These findings reveal, for the first time to our knowledge that besides oat, other grass plants including rice produces protective saponins. Our study provides insight into the role of pathogen-mediated metabolomics reprogramming in host immunity. The correlation between Bayogenin 3-O-Cellobioside levels and blast resistance suggests that engineering saponin expression in cereal crops represents attractive and sustainable disease management.

Small GTPase Rab7-mediated FgAtg9 trafficking is essential for autophagy-dependent development and pathogenicity in Fusarium graminearum.

Fusarium graminearum is a fungal pathogen that causes Fusarium head blight (FHB) in wheat and barley. Autophagy is a highly conserved vacuolar degradation pathway essential for cellular homeostasis in which Atg9 serves as a multispanning membrane protein important for generating membranes for the formation of phagophore assembly site. However, the mechanism of autophagy or autophagosome formation in phytopathogens awaits further clarifications. In this study, we identified and characterized the Atg9 homolog (FgAtg9) in F. graminearum by live cell imaging, biochemical and genetic analyses. We find that GFP-FgAtg9 localizes to late endosomes and trans-Golgi network under both nutrient-rich and nitrogen starvation conditions and also show its dynamic actin-dependent trafficking in the cell. Further targeted gene deletion of FgATG9 demonstrates that it is important for growth, aerial hyphae development, and pathogenicity in F. graminearum. Furthermore, the deletion mutant (ΔFgatg9) shows severe defects in autophagy and lipid metabolism in response to carbon starvation. Interestingly, small GTPase FgRab7 is found to be required for the dynamic trafficking of FgAtg9, and co-immunoprecipitation (Co-IP) assays show that FgAtg9 associates with FgRab7 in vivo. Finally, heterologous complementation assay shows that Atg9 is functionally conserved in F. graminearum and Magnaporthe oryzae. Taken together, we conclude that FgAtg9 is essential for autophagy-dependent development and pathogenicity of F. graminearum, which may be regulated by the small GTPase FgRab7.

Fusarium oxysporum f. sp. lycopersici C2H2 transcription factor FolCzf1 is required for conidiation, fusaric acid production, and early host infection.

The soil-borne, asexual fungus Fusarium oxysporum f.sp. lycopersici (Fol) is a causal agent of tomato wilt disease. The infection process of Fol comprises root recognition, adhesion, penetration, colonization of the root cortex and hyphal proliferation within the xylem vessels, which are under the regulation of virulence-involved transcription factors (TFs). In this study, we identified a gene, designated FolCZF1, which encodes a C2H2 TF in Fol. The homologs of FolCzf1 are also known to affect pathogenicity in F. graminearum and Magnaporthe oryzae on wheat and rice, respectively. We learned that FolCZF1 transcript level is upregulated in conidia and early host infection stage, which led us to hypothesize that FolCzf1 is associated with early host infection in Fol. The FolCZF1 deletion mutant (ΔFolCZF1) exhibited defects in growth rate, conidiation, conidia morphology and a complete loss of virulence on tomato root. Further microscopic observation showed that ΔFolCZF1 can penetrate the root but the primary infection hypha cannot extend its colonization inside the host tissue, suggesting that FolCzf1 TF plays an important role in early infection. Fusaric acid, a secondary metabolite produced by Fusarium species, is suggested as a virulence factor in many crop diseases. We found that FolCzf1 plays a critical role in fusaric acid production by regulating the expression of fusaric acid biosynthesis genes. In summary, FolCzf1 is required for conidiation, secondary metabolism, and early host infection in Fol, and we propose that homologs of FolCzf1 are required for early parasitic growth in other plant pathogenic filamentous fungi.

Disruption of putative short-chain acyl-CoA dehydrogenases compromised free radical scavenging, conidiogenesis, and pathogenesis of Magnaporthe oryzae.

Short-chain acyl-CoA dehydrogenase (Scad) mediated ß-oxidation serves as the fastest route for generating essential energies required to support the survival of organisms under stress or starvation. In this study, we identified three putative SCAD genes in the genome of the globally destructive rice blast pathogen Magnaporthe oryzae, named as MoSCAD1, MoSCAD2, and MoSCAD3. To elucidate their function, we deployed targeted gene deletion strategy to investigate individual and the combined influence of MoSCAD genes on growth, stress tolerance, conidiation and pathogenicity of the rice blast fungus. First, localization and co-localization results obtained from this study showed that MoScad1 localizes to the endoplasmic reticulum (ER), MoScad2 localizes exclusively to the mitochondria while MoScad3 partially localizes to the mitochondria and peroxisome at all developmental stages of M. oryzae. Results obtained from this investigation showed that the deletion of MoSCAD1 and MoSCAD2 caused a minimal but significant reduction in the growth of ΔMoscad1 and ΔMoscad2 strains, while, growth characteristics exhibited by the ΔMoscad3 strain was similar to the wild-type strain. Furthermore, we observed that deletion of MoSCAD2 resulted in drastic reduction in conidiation, delayed conidia germination, triggered the development of abnormal appressorium and suppressed host penetration and colonization efficiencies of the ΔMoscad1 strain. This study provides first material evidence confirming the possible existence of ER ß-oxidation pathway in M. oryzae. We also infer that mitochondria ß-oxidation rather than peroxisomal and ER ß-oxidation play an essential role in the vegetative growth, conidiation, appressorial morphogenesis and progression of pathogenesis in M. oryzae.

Isopropylmalate isomerase MoLeu1 orchestrates leucine biosynthesis, fungal development, and pathogenicity in Magnaporthe oryzae.

The biosynthesis of branched-chain amino acids (BCAAs) is conserved in fungi and plants, but not in animals. The Leu1 gene encodes isopropylmalate isomerase that catalyzes the conversion of α-isopropylmalate into ß-isopropylmalate in the second step of leucine biosynthesis in yeast. Here, we identified and characterized the functions of MoLeu1, an ortholog of yeast Leu1 in the rice blast fungus Magnaporthe oryzae. The transcriptional level of MoLEU1 was increased during conidiation and in infectious stages. Cellular localization analysis indicated that MoLeu1 localizes to the cytoplasm at all stages of fungal development. Targeted gene deletion of MoLEU1 led to leucine auxotrophy, and phenotypic analysis of the generated ∆Moleu1 strain revealed that MoLeu1-mediated leucine biosynthesis was required for vegetative growth, asexual development, and pathogenesis of M. oryzae. We further observed that invasive hyphae produced by the ∆Moleu1 strain were mainly limited to the primary infected host cells. The application of exogenous leucine fully restored vegetative growth and partially restored conidiation as well as pathogenicity defects in the ∆Moleu1 strain. In summary, our results suggested that MoLeu1-mediated leucine biosynthesis crucially promotes vegetative growth, conidiogenesis, and pathogenicity of M. oryzae. This study helps unveil the regulatory mechanisms that are essential for infection-related morphogenesis and pathogenicity of the rice blast fungus.

Comparative genomic analysis revealed rapid differentiation in the pathogenicity-related gene repertoires between Pyricularia oryzae and Pyricularia penniseti isolated from a Pennisetum grass.

BACKGROUND: A number of Pyricularia species are known to infect different grass species. In the case of Pyricularia oryzae (syn. Magnaporthe oryzae), distinct populations are known to be adapted to a wide variety of grass hosts, including rice, wheat and many other grasses. The genome sizes of Pyricularia species are typical for filamentous ascomycete fungi [~ 40 Mbp for P. oryzae, and ~ 45 Mbp for P. grisea]. Genome plasticity, mediated in part by deletions promoted by recombination between repetitive elements [Genome Res 26:1091-1100, 2016, Nat Rev Microbiol 10:417-430,2012] and transposable elements [Annu Rev Phytopathol 55:483-503,2017] contributes to host adaptation. Therefore, comparisons of genome structure of individual species will provide insight into the evolution of host specificity. However, except for the P. oryzae subgroup, little is known about the gene content or genome organization of other Pyricularia species, such as those infecting Pennisetum grasses. RESULTS: Here, we report the genome sequence of P. penniseti strain P1609 isolated from a Pennisetum grass (JUJUNCAO) using PacBio SMRT sequencing technology. Phylogenomic analysis of 28 Magnaporthales species and 5 non-Magnaporthales species indicated that P1609 belongs to a Pyricularia subclade, which is genetically distant from P. oryzae. Comparative genomic analysis revealed that the pathogenicity-related gene repertoires had diverged between P1609 and the P. oryzae strain 70-15, including the known avirulence genes, other putative secreted proteins, as well as some other predicted Pathogen-Host Interaction (PHI) genes. Genomic sequence comparison also identified many genomic rearrangements relative to P. oryzae. CONCLUSION: Our results suggested that the genomic sequence of the P. penniseti P1609 could be a useful resource for the genetic study of the Pennisetum-infecting Pyricularia species and provide new insight into evolution of pathogen genomes during host adaptation.

FgSec2A, a guanine nucleotide exchange factor of FgRab8, is important for polarized growth, pathogenicity and deoxynivalenol production in Fusarium graminearum.

Sec4/Rab8 is one of the well-studied members of the Rab GTPase family, previous studies have shown that Sec4/Rab8 crucially promotes the pathogenesis of phytopathogens, but the upstream regulators of Rab8 are still unknown. Here, we have identified two Sec2 homologues FgSec2A and FgSec2B in devastating fungal pathogen Fusarium graminearum and investigated their functions and interactions with FgRab8 by live-cell imaging, genetic and functional analyses. Yeast two-hybrid assay shows that FgSec2A specifically interacts with FgRab8DN(N123I) and itself. Importantly, FgSec2A is required for growth, conidiation, DON production and virulence of F. graminearum. Live-cell imaging shows that FgSec2A and FgSec2B are both localized to the tip region of hyphae and conidia. Both N-terminal region and Sec2 domain of FgSec2A are essential for its function, but not for localization, whereas the C-terminal region is important for its polarized localization. Furthermore, constitutively active FgRab8CA(Q69L) partially rescues the defects of ΔFgsec2A. Consistently, FgSec2A is required for the polarized localization of FgRab8. Finally, FgSec2A and FgSec2B show partial functions, but FgSec2A does not interact and co-localize with FgSec2B. Taken together, these results indicate that FgSec2A acts as a FgRab8 guanine nucleotide exchange factor and is necessary for polarized growth, DON production and pathogenicity in F. graminearum.

Importance of a Laccase Gene (Lcc1) in the Development of Ganoderma tsugae.

In this study, a novel laccase gene (Lcc1) from Ganoderma tsugae was isolated and its functions were characterized in detail. The results showed that Lcc1 has the highest expression activity during mycelium development and fruit body maturation based on the analysis of Lcc1 RNA transcripts at different developmental stages of G. tsugae. To investigate the exact contribution of Lcc1 to mycelium and fruit body development in G. tsugae, Lcc1 transgenic strains were constructed by targeted gene replacement and over-expression approaches. The results showed that the lignin degradation rate in Lcc1 deletion mutant was much lower than the degradation efficiency of the wild-type (WT), over-expression and rescue strains. The lignin degradation activity of G. tsugae is dependent on Lcc1 and the deletion of Lcc1 exerted detrimental influences on the development of mycelium branch. Furthermore, the study uncovered that Lcc1 deletion mutants generated much shorter pale grey fruit bodies, suggesting that Lcc1 contributes directly to pigmentation and stipe elongation during fruit body development in G. tsugae. The information obtained in this study provides a novel and mechanistic insight into the specific role of Lcc1 during growth and development of G. tsugae.

Methylmalonate-semialdehyde dehydrogenase mediated metabolite homeostasis essentially regulate conidiation, polarized germination and pathogenesis in Magnaporthe oryzae.

Plants generate multitude of aldehydes under abiotic and biotic stress conditions. Ample demonstrations have shown that rice-derived aldehydes enhance the resistance of rice against the rice-blast fungus Magnaporthe oryzae. However, how the fungal pathogen nullifies the inhibitory effects of host aldehydes to establish compatible interaction remains unknown. Here we identified and evaluated the in vivo transcriptional activities of M. oryzae aldehyde dehydrogenase (ALDH) genes. Transcriptional analysis of M. oryzae ALDH genes revealed that the acetylating enzyme Methylmalonate-Semialdehyde Dehydrogenase (MoMsdh/MoMmsdh) elevated activities during host invasion and colonization of the fungus. We further examined the pathophysiological importance of MoMSDH by deploying integrated functional genetics, and biochemical approaches. MoMSDH deletion mutant ΔMomsdh exhibited germination defect, hyper-branching of germ tube and failed to form appressoria on hydrophobic and hydrophilic surface. The MoMSDH disruption caused accumulation of small branch-chain amino acids, pyridoxine and AMP/cAMP in the ΔMomsdh mutant and altered Spitzenkörper organization in the conidia. We concluded that MoMSDH contribute significantly to the pathogenesis of M. oryzae by regulating the mobilization of Spitzenkörper during germ tube morphogenesis, appressoria formation by acting as metabolic switch regulating small branch-chain amino acids, inositol, pyridoxine and AMP/cAMP homeostasis.

WD40-repeat protein MoCreC is essential for carbon repression and is involved in conidiation, growth and pathogenicity of Magnaporthe oryzae.

Carbon catabolite repression (CCR) is a common regulatory mechanism used by microorganisms to prioritize use of a preferred carbon source (usually glucose). The CreC WD40-repeat protein is a major component of the CCR pathway in Aspergillus nidulans. To clarify the function of the CreC ortholog from Magnaporthe oryzae in regulating gene expression important for pathogenesis, MoCreC was identified and genetically characterized. The vegetative growth rate of the MoCreC deletion mutant on various carbon sources was reduced. The MoCreC mutant produced fewer conidia and with about 60% of conidia having septation defects. Appressorium formation was impaired in the MoCreC mutant. Although some appressoria of the mutant could penetrate the leaf surface successfully, the efficiency of penetration and invasive growth of infection hyphae was reduced, resulting in attenuated virulence toward host plants. The CCR was defective as the mutant was more sensitive to allyl alcohol in the presence of glucose, and 2-deoxyglucose was unable to fully repress utilization of secondary carbon sources. qRT-PCR results indicated that the genes encoding cell wall degradation enzymes, such as ß-glucosidase, feruloyl esterase and exoglucanase, were upregulated in MoCreC mutant. Taken together, we conclude that MoCreC is a major regulator of CCR and plays significant roles in regulating growth, conidiation, and pathogenicity of M. oryzae.

Two different subcellular-localized Acetoacetyl-CoA acetyltransferases differentiate diverse functions in Magnaporthe oryzae.

The mevalonate pathway is an efficient biosynthesis pathway that yields isoprenoids for promoting different crucial cellular functions, including ergosterol synthesis and growth regulation. Acetoacetyl-CoA acetyltransferase (EC2.3.1.9) is the first major catalytic enzyme constituting the mevalonate pathway and catalyzes the transformation of Acetoacetyl-CoA from two molecules of acetyl-CoA enroute ergosterol production in fungi. We identified two homologous genes encoding Acetoacetyl-CoA acetyltransferase (MoAcat1 and MoAcat2) in Magnaporthe oryzae, the rice blast fungus. Phylogenetic analysis indicates these two genes have different evolutionary history. We subsequently, conducted targeted gene deletion using homologous recombination technology to ascertain the unique roles of the two MoAcat homologues during the fungal morphogenesis and pathogenesis. The findings from our investigations showed that the activity of MoAcat1 promoted virulence in the rice blast fungus as such, the ΔMoacat1 mutants generated exhibited defect in virulence, whilst ΔMoacat1 mutants did not portray growth defects. ΔMoacat2 mutants on the other hand were characterized by reduction in growth and virulence. Furthermore, MoAcat1 and MoAcat2 showed different expression patterns and subcellular localizations in M. oryzae. From our investigations we came to the conclusion that, different subcellular localization contributes to the diverse functions of MoAcat1 and MoAcat2, which helps the successful establishment of blast disease by promoting efficient development of cell morphology and effective colonization of host tissue.

The CsPbs2-interacting protein oxalate decarboxylase CsOxdC3 modulates morphosporogenesis, virulence, and fungicide resistance in Colletotrichum siamense.

The HOG MAPK pathway mediates diverse cellular and physiological processes, including osmoregulation and fungicide sensitivity, in phytopathogenic fungi. However, the molecular mechanisms underlying HOG MAPK pathway-associated stress homeostasis and pathophysiological developmental events are poorly understood. Here, we demonstrated that the oxalate decarboxylase CsOxdC3 in Colletotrichum siamense interacts with the protein kinase kinase CsPbs2, a component of the HOG MAPK pathway. The expression of the CsOxdC3 gene was significantly suppressed in response to phenylpyrrole and tebuconazole fungicide treatments, while that of CsPbs2 was upregulated by phenylpyrrole and not affected by tebuconazole. We showed that targeted gene deletion of CsOxdC3 suppressed mycelial growth, reduced conidial length, and triggered a marginal reduction in the sporulation characteristics of the ΔCsOxdC3 strains. Interestingly, the ΔCsOxdC3 strain was significantly sensitive to fungicides, including phenylpyrrole and tebuconazole, while the CsPbs2-defective strain was sensitive to tebuconazole but resistant to phenylpyrrole. Additionally, infection assessment revealed a significant reduction in the virulence of the ΔCsOxdC3 strains when inoculated on the leaves of rubber tree (Hevea brasiliensis). From these observations, we inferred that CsOxdC3 crucially modulates HOG MAPK pathway-dependent processes, including morphogenesis, stress homeostasis, fungicide resistance, and virulence, in C. siamense by facilitating direct physical interactions with CsPbs2. This study provides insights into the molecular regulators of the HOG MAPK pathway and underscores the potential of deploying OxdCs as potent targets for developing fungicides.

Transposable elements impact the population divergence of rice blast fungus Magnaporthe oryzae.

Dynamic transposition of transposable elements (TEs) in fungal pathogens has significant impact on genome stability, gene expression, and virulence to the host. In Magnaporthe oryzae, genome plasticity resulting from TE insertion is a major driving force leading to the rapid evolution and diversification of this fungus. Despite their importance in M. oryzae population evolution and divergence, our understanding of TEs in this context remains limited. Here, we conducted a genome-wide analysis of TE transposition dynamics in the 11 most abundant TE families in M. oryzae populations. Our results show that these TEs have specifically expanded in recently isolated M. oryzae rice populations, with the presence/absence polymorphism of TE insertions highly concordant with population divergence on Geng/Japonica and Xian/Indica rice cultivars. Notably, the genes targeted by clade-specific TEs showed clade-specific expression patterns and are involved in the pathogenic process, suggesting a transcriptional regulation of TEs on targeted genes. Our study provides a comprehensive analysis of TEs in M. oryzae populations and demonstrates a crucial role of recent TE bursts in adaptive evolution and diversification of the M. oryzae rice-infecting lineage. IMPORTANCE: Magnaporthe oryzae is the causal agent of the destructive blast disease, which caused massive loss of yield annually worldwide. The fungus diverged into distinct clades during adaptation toward the two rice subspecies, Xian/Indica and Geng/Japonica. Although the role of TEs in the adaptive evolution was well established, mechanisms underlying how TEs promote the population divergence of M. oryzae remain largely unknown. In this study, we reported that TEs shape the population divergence of M. oryzae by differentially regulating gene expression between Xian/Indica-infecting and Geng/Japonica-infecting populations. Our results revealed a TE insertion-mediated gene expression adaption that led to the divergence of M. oryzae population infecting different rice subspecies.

A New Insight into 6-Pentyl-2H-pyran-2-one against Peronophythora litchii via TOR Pathway.

The litchi downy blight disease of litchi caused by Peronophythora litchii accounts for severe losses in the field and during storage. While ample quantitative studies have shown that 6-pentyl-2H-pyran-2-one (6PP) possesses antifungal activities against multiple plant pathogenic fungi, the regulatory mechanisms of 6PP-mediated inhibition of fungal pathogenesis and growth are still unknown. Here, we investigated the potential molecular targets of 6PP in the phytopathogenic oomycetes P. litchii through integrated deployment of RNA-sequencing, functional genetics, and biochemical techniques to investigate the regulatory effects of 6PP against P. litchii. Previously we demonstrated that 6PP exerted significant oomyticidal activities. Also, comparative transcriptomic evaluation of P. litchii strains treated with 6PP Revealed significant up-regulations in the expression profile of TOR pathway-related genes, including PlCytochrome C and the transcription factors PlYY1. We also noticed that 6PP treatment down-regulated putative negative regulatory genes of the TOR pathway, including PlSpm1 and PlrhoH12 in P. litchii. Protein-ligand binding analyses revealed stable affinities between PlYY1, PlCytochrome C, PlSpm1, PlrhoH12 proteins, and the 6PP ligand. Phenotypic characterization of PlYY1 targeted gene deletion strains generated in this study using CRISPR/Cas9 and homologous recombination strategies significantly reduced the vegetative growth, sporangium, encystment, zoospore release, and pathogenicity of P. litchii. These findings suggest that 6PP-mediated activation of PlYY1 expression positively regulates TOR-related responses and significantly influences vegetative growth and the virulence of P. litchii. The current investigations revealed novel targets for 6PP and underscored the potential of deploying 6PP in developing management strategies for controlling the litchi downy blight pathogen.

Cytoplasmic dynein1 intermediate-chain2 regulates cellular trafficking and physiopathological development in Magnaporthe oryzae.

The cytoplasmic dynein 1, a minus end-directed motor protein, is an essential microtubule-based molecular motor that mediates the movement of molecules to intracellular destinations in eukaryotes. However, the role of dynein in the pathogenesis of Magnaporthe oryzae is unknown. Here, we identified cytoplasmic dynein 1 intermediate-chain 2 genes in M. oryzae and functionally characterized it using genetic manipulations, and biochemical approaches. We observed that targeted the deletion of MoDYNC1I2 caused significant vegetative growth defects, abolished conidiation, and rendered the ΔModync1I2 strains non-pathogenic. Microscopic examinations revealed significant defects in microtubule network organization, nuclear positioning, and endocytosis ΔModync1I2 strains. MoDync1I2 is localized exclusively to microtubules during fungal developmental stages but co-localizes with the histone OsHis1 in plant nuclei upon infection. The exogenous expression of a histone gene, MoHis1, restored the homeostatic phenotypes of ΔModync1I2 strains but not pathogenicity. These findings could facilitate the development of dynein-directed remedies for managing the rice blast disease.

A Putative P-Type ATPase Regulates the Secretion of Hydrolytic Enzymes, Phospholipid Transport, Morphogenesis, and Pathogenesis in Phytophthora capsici.

Phytophthora capsici is an important plant pathogenic oomycete with multiple hosts. The P4-ATPases, aminophospholipid translocases (APTs), play essential roles in the growth and pathogenesis of fungal pathogens. However, the function of P4-ATPase in P. capsici remains unclear. This study identified and characterized PcApt1, a P4-ATPase Drs2 homolog, in P. capsici. Deletion of PcAPT1 by CRISPR/Cas9 knock-out strategy impaired hyphal growth, extracellular laccase activity. Cytological analyses have shown that PcApt1 participates in phosphatidylserine (PS) transport across the plasma membrane. Also, we showed that targeted deletion of PcAPT1 triggered a significant reduction in the virulence of P. capsici. Secretome analyses have demonstrated that secretion of hydrolytic enzymes decreased considerably in the PcAPT1 gene deletion strains compared to the wild-type. Overall, our results showed that PcApt1 plays a pivotal role in promoting morphological development, phospholipid transport, secretion of hydrolytic enzymes, and the pathogenicity of the polycyclic phytopathogenic oomycete P. capsici. This study underscores the need for comprehensive evaluation of subsequent members of the P-type ATPase family to provide enhanced insights into the dynamic contributions to the pathogenesis of P. capsici and their possible deployment in the formulation of effective control strategies.

Magnaporthe oryzae Chloroplast Targeting Endo-ß-1,4-Xylanase I MoXYL1A Regulates Conidiation, Appressorium Maturation and Virulence of the Rice Blast Fungus.

Endo-ß-1,4-Xylanases are a group of extracellular enzymes that catalyze the hydrolysis of xylan, a principal constituent of the plant primary cell wall. The contribution of Endo-ß-1,4-Xylanase I to both physiology and pathogenesis of the rice blast fungus M. oryzae is unknown. Here, we characterized the biological function of two endoxylanase I (MoXYL1A and MoXYL1B) genes in the development of M. oryzae using targeted gene deletion, biochemical analysis, and fluorescence microscopy. Phenotypic analysis of ∆Moxyl1A strains showed that MoXYL1A is required for the full virulence of M. oryzae but is dispensable for the vegetative growth of the rice blast fungus. MoXYL1B, in contrast, did not have a clear role in the infectious cycle but has a critical function in asexual reproduction of the fungus. The double deletion mutant was severely impaired in pathogenicity and virulence as well as asexual development. We found that MoXYL1A deletion compromised appressorium morphogenesis and function, leading to failure to penetrate host cells. Fluorescently tagged MoXYL1A and MoXYL1B displayed cytoplasmic localization in M. oryzae, while analysis of MoXYL1A-GFP and MoXYL1B-GFP in-planta revealed translocation and accumulation of these effector proteins into host cells. Meanwhile, sequence feature analysis showed that MoXYL1A possesses a transient chloroplast targeting signal peptide, and results from an Agrobacterium infiltration assay confirmed co-localization of MoXYL1A-GFP with ChCPN10C-RFP in the chloroplasts of host cells. MoXYL1B, accumulated to the cytoplasm of the host. Taken together, we conclude that MoXYL1A is a secreted effector protein that likely promotes the virulence of M. oryzae by interfering in the proper functioning of the host chloroplast, while the related xylanase MoXYL1B does not have a major role in virulence of M. oryzae.

Translation Initiation Factor eIF4E Positively Modulates Conidiogenesis, Appressorium Formation, Host Invasion and Stress Homeostasis in the Filamentous Fungi Magnaporthe oryzae.

Translation initiation factor eIF4E generally mediates the recognition of the 5'cap structure of mRNA during the recruitment of the ribosomes to capped mRNA. Although the eIF4E has been shown to regulate stress response in Schizosaccharomyces pombe positively, there is no direct experimental evidence for the contributions of eIF4E to both physiological and pathogenic development of filamentous fungi. We generated Magnaporthe oryzae eIF4E (MoeIF4E3) gene deletion strains using homologous recombination strategies. Phenotypic and biochemical analyses of MoeIF4E3 defective strains showed that the deletion of MoeIF4E3 triggered a significant reduction in growth and conidiogenesis. We also showed that disruption of MoeIF4E3 partially impaired conidia germination, appressorium integrity and attenuated the pathogenicity of ΔMoeif4e3 strains. In summary, this study provides experimental insights into the contributions of the eIF4E3 to the development of filamentous fungi. Additionally, these observations underscored the need for a comprehensive evaluation of the translational regulatory machinery in phytopathogenic fungi during pathogen-host interaction progression.

eIF3k Domain-Containing Protein Regulates Conidiogenesis, Appressorium Turgor, Virulence, Stress Tolerance, and Physiological and Pathogenic Development of Magnaporthe oryzae Oryzae.

The eukaryotic translation initiation factor 3 (eIF3) complex consists of essential and non-essential sub-complexes. Non-essential eIF3 complex subunits, such as eIF3e, eIF3j, eIF3k, and eIF3l, modulate stress tolerance and enhance the lifespan of Neurospora crassa and Caenorhabditis elegans. However, there is limited knowledge of the role of the non-essential eIF3 sub-complex in the pathophysiological development of plant fungal pathogens. Here, we deployed genetic and biochemical techniques to explore the influence of a hypothetical protein containing eIF3k domain in Magnaporthe oryzae Oryzae (MoOeIF3k) on reproduction, hyphae morphogenesis, stress tolerance, and pathogenesis. Also, the targeted disruption of MoOeIF3k suppressed vegetative growth and asexual sporulation in ΔMoOeif3k strains significantly. We demonstrated that MoOeIF3k promotes the initiation and development of the rice blast disease by positively regulating the mobilization and degradation of glycogen, appressorium integrity, host penetration, and colonization during host-pathogen interaction. For the first time, we demonstrated that the eIF3k subunit supports the survival of the blast fungus by suppressing vegetative growth and possibly regulating the conversions and utilization of stored cellular energy reserves under starvation conditions. We also observed that the deletion of MoOeIF3k accelerated ribosomal RNA (rRNA) generation in the ΔMoOeif3k strains with a corresponding increase in total protein output. In summary, this study unravels the pathophysiological significance of eIF3k filamentous fungi. The findings also underscored the need to systematically evaluate the individual subunits of the non-essential eIF3 sub-complex during host-pathogen interaction. Further studies are required to unravel the influence of synergetic coordination between translation and transcriptional regulatory machinery on the pathogenesis of filamentous fungi pathogens.