Pathogenicity analysis and comparative genomics reveal the different infection strategies between the generalist Metarhizium anisopliae and the specialist Metarhizium acridum.

BACKGROUND: The fungal genera Metarhizium contain many important multiple species that are used as biocontrol agents and as model organisms for exploring insect-fungal interactions. Metarhizium spp. exhibit different traits of pathogenicity, suggesting that the pathogenesis can be quite distinctive. However, the underlying differences in their pathogenesis remain poorly understood. RESULTS: Pathogenicity analysis showed that Metarhizium anisopliae (strain CQMa421) displayed higher virulence against oriental migratory locusts, Locusta migratoria manilensis (Meyen), than the acridid-specific specie Metarhizium acridum (strain CQMa102). Relative to M. acridum, M. anisopliae possessed a higher conidial hydrophobicity, increased ability to penetrate the host, accelerated growth under hypoxia and enhanced ability for the utilization of different carbon sources. Different distributions of carbohydrate epitopes at cell wall surface of M. anisopliae might also contribute to successful evasion of host immune defenses. Comparative genomics showed that M. anisopliae has 98 more virulence-related secreted proteins (133) than M. acridum (35), which can be functionally classified as hydrolases, virulence effectors, cell wall degradation and stress tolerance-related proteins, and helpful to the cuticle penetration and host internal environment adaption. In addition, differences in genomic clusters specifically related to secondary metabolites, including the clusters of Indole-NRPS hybrid, T1PKS-NRPS like hybrid, Betalactone, Fungal-Ripp and NRPS-Terpene hybrid, may lead to differences in core virulence-related secondary metabolite genes in M. acridum (18) and M. anisopliae (36). CONCLUSION: The comparative study provided new insights into the different infection strategies between M. anisopliae and M. acridum, and further facilitate the identification of virulence-related genes for the improvement of mycoinsecticides. © 2023 Society of Chemical Industry.

An insect chemosensory protein facilitates locust avoidance to fungal pathogens via recognition of fungal volatiles.

Locusts (Locusta migratoria) are one of the most destructive insect pests worldwide. Entomopathogenic fungi can infect and kill locusts, with Metarhizium acridum having evolved as a specialized acridid pathogen. However, locusts have evolved countermeasures to limit or avoid microbial pathogens, although the underlying molecular mechanisms behind these defenses remain obscure. Here, we demonstrate that L. migratoria exhibit avoidance behaviors towards M. acridum contaminated food via recognition of fungal volatiles, with locust perception of the volatile mediated by the LmigCSP60 chemosensory protein. RNAi-knockdown of LmigCSP60 lowered locust M. acridum avoidance behavior and increased infection and mortality. The fungal volatile, 2-phenylethanol (PEA), was identified to participate in locust behavioral avoidance. RNAi-knockdown of LmigCSP60 reduced antennal electrophysiological responses to PEA and impaired locust avoidance to the compound. Purified LmigCSP60 was able to bind a set of fungal volatiles including PEA. Furthermore, reduction of PEA emission by M. acridum via construction of a targeted gene knockout mutant of the alcohol dehydrogenase gene (ΔMaAdh strain) that contributes to PEA production reduced locust avoidance behavior towards the pathogen. These findings identify an olfactory circuit used by locusts to detect and avoid potential microbial pathogens before they are capable of initiating infection and highlight behavioral and olfactory adaptations affecting the co-evolution of host-pathogen interactions.

A virus induces alterations in root morphology while exerting minimal effects on the rhizosphere and endosphere microorganisms in rice.

The highly destructive southern rice black-streaked dwarf virus (SRBSDV) causes significant losses in rice production. To understand its impact on rice root, we studied fibrous root development and root microbiota variation (rhizosphere and endosphere) after SRBSDV infection. SRBSDV infection reduced the number and length of fibrous roots in rice. Interestingly, the rhizosphere had higher bacterial diversity and abundance at the initial (0 days) and 30-day postinfection stages, while 30-day-old roots showed increased diversity and abundance. However, there were no significant differences in microbiota diversity between infected and noninfected rice plants. The major rhizosphere microbiota included Proteobacteria, Bacteroidota, Acidobacteriota, and Planctomycetota, comprising about 80% of the community. The endosphere was dominated by Proteobacteria and Cyanobacteria, constituting over 90%, with Bacteroidota as the next most prominent group. Further, we identified differentially expressed genes related to plant-pathogen interactions, plant hormone signal, and ABC transporters, potentially affecting root morphology. Notably, specific bacteria (e.g. Inquilinus and Actinoplanes) showed correlations with these pathways. In conclusion, SRBSDV primarily influences root growth through host metabolism, rather than exerting direct effects on the root microbiota. These insights into the interactions among the pathogen, rice plant, and associated microbiota could have implications for managing SRBSDV's detrimental effects on rice production.

Recent Advancements in Pathogenic Mechanisms, Applications and Strategies for Entomopathogenic Fungi in Mosquito Biocontrol.

Fungal diseases are widespread among insects and play a crucial role in naturally regulating insect populations. Mosquitoes, known as vectors for numerous infectious diseases, pose a significant threat to human health. Entomopathogenic fungi (EPF) have emerged as highly promising alternative agents to chemical mosquitocides for controlling mosquitoes at all stages of their life cycle due to their unique infection pathway through direct contact with the insect's cuticle. In recent years, significant advancements have been made in understanding the infection pathways and pathogenic mechanisms of EPF against mosquitoes. Various strategies involving the use of EPF alone or combinations with other approaches have been employed to target mosquitoes at various developmental stages. Moreover, the application of genetic technologies in fungi has opened up new avenues for enhancing the mosquitocidal efficacy of EPF. This review presents a comprehensive summary of recent advancements in our understanding the pathogenic mechanisms of EPF, their applications in mosquito management, and the combination of EPF with other approaches and employment of transgenic technologies. The biosafety concerns associated with their use and the corresponding approaches are also discussed. The recent progress suggests that EPF have the potential to serve as a future biorational tool for controlling mosquito vectors.

Insights into dendritic cell maturation during infection with application of advanced imaging techniques.

Dendritic cells (DCs) are crucial for the initiation and regulation of adaptive immune responses. When encountering immune stimulus such as bacterial and viral infection, parasite invasion and dead cell debris, DCs capture antigens, mature, acquire immunostimulatory activity and transmit the immune information to naïve T cells. Then activated cytotoxic CD8+ T cells directly kill the infected cells, while CD4+ T helper cells release cytokines to aid the activity of other immune cells, and help B cells produce antibodies. Thus, detailed insights into the DC maturation process are necessary for us to understand the working principle of immune system, and develop new medical treatments for infection, cancer and autoimmune disease. This review summarizes the DC maturation process, including environment sensing and antigen sampling by resting DCs, antigen processing and presentation on the cell surface, DC migration, DC-T cell interaction and T cell activation. Application of advanced imaging modalities allows visualization of subcellular and molecular processes in a super-high resolution. The spatiotemporal tracking of DCs position and migration reveals dynamics of DC behavior during infection, shedding novel lights on DC biology.

Tetracarboxylic acid transporter regulates growth, conidiation, and carbon utilization in Metarhizium acridum.

Carbon sources and their utilization are vital for fungal growth and development. C4-dicarboxylic acids are important carbon and energy sources that function as intermediate products of the tricarboxylic acid cycle. Transport and regulation of C4-dicarboxylic acid uptake are mainly dependent on tetracarboxylic acid transporters (Dcts) in many microbes, although the roles of Dct genes in fungi have only been partially characterized. Here, we report on the functions of two Dct genes (Dct1 and Dct2) in the entomopathogenic fungus Metarhizium acridum. Our data showed that loss of the MaDct1 gene affected utilization of tetracarboxylic acids and other carbon sources. ΔMaDct1 mutants showed larger colony sizes with extensive mycelial growth but were delayed in conidiation with decreased conidia yield as compared to the wild-type parental strain. On the nutrient-deficient medium, SYA, the wild-type strain produced microcycle conidia, whereas the ΔMaDct1 mutant produced (normal) aerial conidia. In addition, ΔMaDct1 had decreased tolerance to cell wall perturbing agents, but increased tolerances to UV-B radiation and osmotic stress. Insect bioassays indicated that loss of MaDct1 did not affect pathogenicity. In contrast, no distinct phenotypic change was observed for the MaDct2 mutant in terms of growth and biocontrol characteristics. Transcriptomic profiling between wild type and ΔMaDct1 showed that differentially expressed genes were enriched in carbohydrate and amino acid metabolism, transport and catabolism, and signal transduction. These results demonstrate that MaDct1 regulates the conidiation pattern shift and mycelial growth by affecting utilization of carbon sources. These findings are helpful for better understanding the effect of intermediates of carbon metabolism on fungal growth and conidiation. KEY POINTS: • MaDct1 influences fungal growth and conidiation by affecting carbon source utilization. • MaDct1 regulates conidiation pattern shift under nutrient deficiency condition. • MaDct1 is involved in stress tolerance and has no effect on virulence. • MaDct2 has no effect on growth and biocontrol characteristic.

Arginine metabolism governs microcycle conidiation by changing nitric oxide content in Metarhizium acridum.

Microcycle conidiation commonly exists in filamentous fungi and has great potential for mass production of mycoinsecticides. L-Arginine metabolism is essential for conidiation and conditional growth and virulence, but its role in microcycle conidiation has not been explored. Here, a unique putative arginase (MaAGA) was characterized in the entomopathogenic fungus Metarhizium acridum. Conidial germination and thermotolerance were facilitated by the disruption of MaAGA. Despite little impact on fungal growth and virulence, the disruption resulted in normal conidiation after a 60-h incubation on microcycle conidiation medium (SYA) under normal culture conditions. In the MaAGA-disruption mutant (ΔMaAGA), intracellular arginine accumulation was sharply increased. Replenishment of the direct metabolites of arginase, namely ornithine and/or urea, was unable to restore the disruption mutant's microcycle conidiation on SYA. Interestingly, nitric oxide synthase (NOS) activity and nitric oxide (NO) levels of the ΔMaAGA strain were markedly decreased in the 60-h-old SYA cultures. Finally, adding Nω-nitro-L-arginine, an inhibitor of NOS, into the SYA converted the microcycle conidiation of the wild-type strain to normal conidiation. In contrast, adding sodium nitroprusside, an NO donor, into the SYA recovered the mutant's microcycle conidiation. The results indicate that arginine metabolism controls microcycle conidiation by changing the content of NO. KEY POINTS: • The MaAGA-disruption led to normal conidiation on microcycle conidiation medium SYA. • Nitric oxide (NO) level of the ΔMaAGA strain was markedly decreased. • Adding an NO donor into the SYA recovered the microcycle conidiation of ΔMaAGA.

An odorant binding protein is involved in counteracting detection-avoidance and Toll-pathway innate immunity.

INTRODUCTION: Odorant-binding proteins (OBPs) are a class of small molecular weight soluble proteins that exist as expanded gene families in all insects, acting as ligand carriers mediating olfaction and other physiological processes. During fungal infection, a subset of insect OBPs were shown to be differentially expressed. OBJECTIVES: We tested whether the altered expression of insect OBPs during pathogenic infection plays a role in behavioral or immune interactions between insect hosts and their pathogens. METHODS: A wide range of techniques including RNAi-directed knockdown, heterologous protein expression, electrophysiological/behavioral analyses, transcriptomics, gut microbiome analyses, coupled with tandem mass spectrometry ion monitoring, were used to characterize the function of a locust OBP in host behavioral and immune responses. RESULTS: The entomopathogenic fungus Metarhizium anisopliae produces the volatile compound phenylethyl alcohol (PEA) that causes behavioral avoidance in locusts. This is mediated by the locust odorant binding protein 11 (LmOBP11). Expression of LmOBP11 is induced by M. anisopliae infection and PEA treatment. LmOBP11 participates in insect detection of the fungal-produced PEA and avoidance of PEA-contaminated food, but the upregulation of LmOBP11 upon M. anisopliae infection negatively affects the insect immune responses to ultimately benefit successful mycosis by the pathogen. RNAi knockdown of LmOBP11 increases the production of antimicrobial peptides and enhances locust resistance to M. anisopliae infection, while reducing host antennal electrophysiological responses to PEA and locust avoidance of PEA treated food. Also, transcriptomic and gut microbiome analyses reveal microbiome dysbiosis and changes in host genes involved in behavior and immunity. These results are consistent with the elevated expression of LmOBP11 leading to enhanced volatile detection and suppression of immune responses. CONCLUSION: These findings suggest a crosstalk between olfaction and immunity, indicating manipulation of host OBPs as a novel target exploited by fungal pathogens to alter immune activation and thus promote the successful infection of the host.

Comparative analysis of gut microbiota and immune genes linked with the immune system of wild and captive Spodoptera frugiperda (Lepidoptera: Noctuidae).

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is one of the most highly polyphagous invasive pests causing serious damage to maize crops in China. However, little is known about the gut immune responses to the environment, particularly along the migration routes in Jianghuai, China, throughout the autumn and winter. In this study, high-throughput sequencing and real-time quantitative PCR (RT-qPCR) were employed to examine the variations in immune genes and gut microbiome communities between captive and wild fall armyworm populations. Results showed that the diversity and community of the gut's microbes were higher in wild populations, and the average weighted UniFrac distance between bacterial taxa varied. A wide variety of immune genes were more abundant in the wild populations than in others. Results indicated that diets and different survival conditions impacted the gut microbiota and immune system of S. frugiperda, which was crucial for environmental adaptation. These differences in gut microbiota and immune responses between wild and captive Fall armyworms are critical for comprehending the symbiotic relationship between microbes, immune genes, and hosts. They also highlight the need for increased focus on developing more effective and environmentally friendly pest control methods.

MaCts1, an Endochitinase, Is Involved in Conidial Germination, Conidial Yield, Stress Tolerances and Microcycle Conidiation in Metarhizium acridum.

Entomopathogenic fungi are promising biocontrol agents of insect-mediated crop damage. Microcycle conidiation has shown great potential in enhancing the conidial yield and quality of entomopathogenic fungi. Homologs of Cts1, an endochitinase of Saccharomyces cerevisiae, participate in cell separation in several fungal spp. and may contribute to the morphological differences that occur during the shift to microcycle conidiation. However, the precise functions of Cts1 in entomopathogenic fungi remain unclear. Herein, the endochitinase gene, MaCts1, was characterized in the model entomopathogen, Metarhizium acridum. A loss of function line for MaCts1 led to a delay of 1 h in the median germination time, a 28% reduction in conidial yield and significant defects in fungal resistances to UV-irradiation (18%) and heat-shock (15%), while fungal tolerances to cell wall stressors, oxidative and hyperosmotic stresses and virulence remained unchanged. The MaCts1-disruption strain displayed typical conidiation on the microcycle conidiation induction medium, SYA. In contrast, deletion of key genes in the morphogenesis-related NDR kinase network (MOR pathway)/regulation of Ace2 and morphogenesis (RAM pathway) did not affect the SYA-induction of microcycle conidiation. This indicates that MaCts1 makes contributions to the microcycle conidiation, which may not be dependent on the MOR/RAM pathway in M. acridum.

The C2H2 Zinc Finger Protein MaNCP1 Contributes to Conidiation through Governing the Nitrate Assimilation Pathway in the Entomopathogenic Fungus Metarhizium acridum.

Zinc finger proteins are an important class of multifunctional regulators. Here, the roles of a C2H2 zinc finger protein MaNCP1 (Metarhizium acridum nitrate-related conidiation pattern shift regulatory factor 1) in nitrogen utilization and conidiation were explored in the entomopathogenic fungus M. acridum. The results showed that MaNCP1-disruption mutant (ΔMaNCP1) impaired the ability to utilize nitrate, ammonium and glutamine and reduced the expression of nitrate assimilation-related genes, suggesting that MaNCP1 was involved in governing nitrogen utilization. In addition, the conidial yield of the ΔMaNCP1 strain, cultured on the microcycle conidiation medium (SYA), was significantly decreased, which could be restored or even enhanced than that of the WT strain through increasing the nitrate content in SYA medium. Further study showed that MaAreA, a core regulator in the nitrogen catabolism repression (NCR) pathway, was a downstream target gene of MaNCP1. Screening the differential expression genes between WT and ΔMaNCP1 strains revealed that the conidial yield of M. acridum regulated by nitrate might be related to NCR pathway on SYA medium. It could be concluded that MaNCP1 contributes to the nitrate assimilation and conidiation, which will provide further insights into the relationship between the nitrogen utilization and conidiation in fungi.

N-terminal zinc fingers of MaNCP1 contribute to growth, stress tolerance, and virulence in Metarhizium acridum.

C2H2 zinc finger proteins (ZFPs) are a class of important transcriptional regulators in eukaryotes involved in multiple biological regulation processes. Here, MaNCP1, a C2H2 ZFP, was functionally characterized in the model entomopathogenic fungus Metarhizium acridum. Deletion of MaNCP1 delayed conidial germination and hyphal growth, decreased the conidial yield and reduced the tolerances to UV-B irradiation and heat-shock. The N-terminal zinc fingers (ZFs) of MaNCP1 made the main contributions to these traits. In addition, disruption of MaNCP1 altered the conidial surface structure and decreased the conidial hydrophobicity. Bioassays showed that the virulence of the MaNCP1-disruption strain (ΔMaNCP1) was reduced in topical inoculation compared to the WT or the mutant complemented strain (CP), and the N-terminal C2H2 ZFs made a major contribution to virulence. Furthermore, the ΔMaNCP1 and C2H2 ZFs deletion mutants (MaNCP1∆N and MaNCP1∆N+C) impaired cuticular penetration. RNA-seq showed that several cuticle-degrading genes were down-regulated in the ΔMaNCP1 background, suggesting that MaNCP1 plays vital roles in regulating insect cuticle penetration. In summary, MaNCP1 affected the growth, stress tolerances and virulence of M. acridum, and the N-terminal C2H2 ZFs played indispensable roles in these important biocontrol traits. These results provide further insights into the functions of C2H2 ZFPs in entomopathogenic fungi.

MaOpy2, a Transmembrane Protein, Is Involved in Stress Tolerances and Pathogenicity and Negatively Regulates Conidial Yield by Shifting the Conidiation Pattern in Metarhizium acridum.

Opy2 is an important membrane-anchored protein upstream of the HOG-MAPK signaling pathway and plays important roles in both the HOG-MAPK and Fus3/Kss1 MAPK. In this study, the roles of MaOpy2 in Metarhizium acridum were systematically elucidated. The results showed that the MaOpy2 disruption significantly reduced fungal tolerances to UV, heat shock and cell-wall-disrupting agents. Bioassays showed that the decreased fungal pathogenicity by topical inoculation mainly resulted from the impaired penetration ability. However, the growth ability of ∆MaOpy2 was enhanced in insect hemolymph. Importantly, MaOpy2 deletion could significantly increase the conidial yield of M. acridum by shifting the conidiation pattern from normal conidiation to microcycle conidiation on the 1/4SDAY medium. Sixty-two differentially expressed genes (DEGs) during the conidiation pattern shift, including 37 up-regulated genes and 25 down-regulated genes in ∆MaOpy2, were identified by RNA-seq. Further analysis revealed that some DEGs were related to conidiation and hyphal development. This study will provide not only the theoretical basis for elucidating the regulation mechanism for improving the conidial yield and quality in M. acridum but also theoretical guidance for the molecular improvement of entomopathogenic fungi.

Transcription Factor Mavib-1 Negatively Regulates Conidiation by Affecting Utilization of Carbon and Nitrogen Source in Metarhizium acridum.

Conidium is the main infection unit and reproductive unit of pathogenic fungi. Exploring the mechanism of conidiation and its regulation contributes to understanding the pathogenicity of pathogenic fungi. Vib-1, a transcription factor, was reported to participate in the conidiation process. However, the regulation mechanism of Vib-1 in conidiation is still unclear. In this study, we analyzed the function of Vib-1 and its regulation mechanism in conidiation through knocking out and overexpression of Vib-1 in entomopathogenic fungus Metarhizium acridum. Results showed that the colonial growth of Mavib-1 disruption mutant (ΔMavib-1) was significantly decreased, and conidiation was earlier compared to wild type (WT), while overexpression of Mavib-1 led to a delayed conidiation especially when carbon or nitrogen sources were insufficient. Overexpression of Mavib-1 resulted in a conidiation pattern shift from microcycle conidiation to normal conidiation on nutrient-limited medium. These results indicated that Mavib-1 acted as a positive regulator in vegetative growth and a negative regulator in conidiation by affecting utilization of carbon and nitrogen sources in M. acridum. Transcription profile analysis demonstrated that many genes related to carbon and nitrogen source metabolisms were differentially expressed in ΔMavib-1 and OE strains compared to WT. Moreover, Mavib-1 affects the conidial germination, tolerance to UV-B and heat stresses, cell wall integrity, conidial surface morphology and conidial hydrophobicity in M. acridum. These findings unravel the regulatory mechanism of Mavib-1 in fungal growth and conidiation, and enrich the knowledge to conidiation pattern shift of filamentous fungi.

Host-Pathogen Interactions between Metarhizium spp. and Locusts.

The progress in research on the interactions between Metarhizium spp. and locusts has improved our understanding of the interactions between fungal infection and host immunity. A general network of immune responses has been constructed, and the pathways regulating fungal pathogenicity have also been explored in depth. However, there have been no systematic surveys of interaction between Metarhizium spp. and locusts. The pathogenesis of Metarhizium comprises conidial attachment, germination, appressorial formation, and colonization in the body cavity of the host locusts. Meanwhile, the locust resists fungal infection through humoral and cellular immunity. Here, we summarize the crucial pathways that regulate the pathogenesis of Metarhizium and host immune defense. Conidial hydrophobicity is mainly affected by the contents of hydrophobins and chitin. Appressorial formation is regulated by the pathways of MAPKs, cAMP/PKA, and Ca2+/calmodulin. Lipid droplets degradation and secreted enzymes contributed to fungal penetration. The humoral response of locust is coordinated by the Toll pathway and the ecdysone. The regulatory mechanism of hemocyte differentiation and migration is elusive. In addition, behavioral fever and density-dependent population immunity have an impact on the resistance of hosts against fungal infection. This review depicts a prospect to help us understand host-pathogen interactions and provides a foundation for the engineering of entomopathogenic fungi and the discovery of insecticidal targets to control insect pests.

The Entomopathogenic Fungus Metarhizium anisopliae Affects Feeding Preference of Sogatella furcifera and Its Potential Targets' Identification.

The rice planthopper Sogatella furcifera is a unique vector of the southern rice black-streaked dwarf virus (SRBSDV). The feeding behavior of S. furcifera should directly affect the diffusion of this virus. In this study, we noted that the infection of Metarhizium anisopliae CQMa421 on S. furcifera disturbed the feeding behavior of this pest to SRBSDV-infected rice, from preference to non-preference. Then, we further investigated the potential targets of M. anisopliae CQMa421 on the feeding behavior of S. furcifera after 0 h, 24 h and 48 h of infection by transcriptomic analysis via Illumina deep sequencing. A total of 93.27 GB of data was collected after sequencing, from which 91,125 unigenes were annotated, including 75 newly annotated genes. There were 1380 vs. 2187 and 137 vs. 106 upregulated and downregulated differentially expressed genes (DEGs) detected at 24 h and 48 h, respectively. The biological functions and associated metabolic processes of these genes were determined with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The results suggested that major of DEGs are involved in energy metabolism, biosynthesis, immune response, the FoxO signaling pathway, the MAPK signaling pathway and apoptosis in response to the fungal infection. Noteworthily, several olfactory-related genes, including odorant receptors and odorant binding proteins, were screened from these differentially expressed genes, which played critical roles in regulating the olfactory behavior of insects. Taken together, these results provide new insights for understanding the molecular mechanisms underlying fungus and host insect interaction, especially for olfactory behavior regulated by fungus.

The carbon catabolite repressor CreA is an essential virulence factor of Metarhizium acridum against Locusta migratoria.

BACKGROUND: CreA has been proved to be a core gene in asexual conidiation in Metarhizium acridum, which regulates the shift of normal conidiation and microcycle conidiation. At present, research on CreA in fungi has focused on carbon source metabolism. There is a lack of research on the effect of CreA in virulence of pathogenic fungi. RESULTS: The virulence of the MaCreA disrupted strain (ΔMaCreA) for Locusta migratoria was lost by topical inoculation bioassay. The formation rate and turgor pressure of the appressoria decreased. Growth of ΔMaCreA in host hemolymph was delayed, and the number of hyphal bodies was significantly reduced. The conidial cell wall of ΔMaCreA became thicker, the mannan content decreased, and the chitin content increased significantly, and it was more sensitive to calcofluor white and Congo Red. α-1,3-Glucan and ß-1,3-glucan are more exposed on the surface of ΔMaCreA conidia than on the wild type. Lmspätzle and Lmcactus, the immune response genes in the host Toll pathway, showed stronger transcriptional activities at the early stage of ΔMaCreA invasion. The phenoloxidase activity assay also showed stronger immunostimulation by ΔMaCreA in vitro. CONCLUSION: The main reasons for the loss of virulence of ΔMaCreA in the topical inoculation were the reduced penetration ability of appressoria, limited growth in hemolymph and stronger insect immunostimulation of ΔMaCreA. © 2022 Society of Chemical Industry.

MaNCP1, a C2H2 Zinc Finger Protein, Governs the Conidiation Pattern Shift through Regulating the Reductive Pathway for Nitric Oxide Synthesis in the Filamentous Fungus Metarhizium acridum.

Asexual sporulation is the most common reproduction mode of fungi. Most filamentous fungi have two conidiation patterns, normal conidiation and microcycle conidiation, which may be regulated by nutritional conditions. Nitrogen source can affect the fungal conidiation pattern, but the regulatory mechanism is not fully understood. In this study, we report a C2H2 zinc finger protein, MaNCP1, which has typical transcription factor characteristics and is screened from the subtractive library regulated by nitrate in the entomopathogenic fungus Metarhizium acridum. MaNCP1 and its N-terminal play critical roles in the conidiation pattern shift. Further study shows that MaNCP1 interacts with MaNmrA, which also contributes to the conidiation pattern shift and is involved in the reductive pathway of nitric oxide (NO) synthesis. Intriguingly, the conidiation pattern of the MaNCP1-disruption strain (ΔMaNCP1) can be restored to microcycle conidiation when grown on the microcycle conidiation medium, SYA, supplemented with NO donor or overexpressing MaNmrA in ΔMaNCP1. Here, we reveal that MaNCP1 governs the conidiation pattern shift through regulating the reductive synthesis of NO by physically targeting MaNmrA in M. acridum. This work provides new mechanistic insights into how changes in nitrogen utilization are linked to the regulation of fungal morphological changes. IMPORTANCE Fungal conidia play important roles in the response to environmental stimuli and evasion of the host immune system. The nitrogen source is one of the main factors affecting shifts in fungal conidiation patterns, but the regulatory mechanism involved is not fully understood. In this work, we report that the C2H2 zinc finger protein, MaNCP1, governs the conidiation pattern shift in M. acridum by targeting the MaNmrA gene, thereby altering the regulation of the reductive pathway for NO synthesis. This work provides further insights into how the nutritional environment can regulate the morphogenesis of filamentous fungi.

MaNsdD regulates conidiation negatively by inhibiting the AbaA expression required for normal conidiation in Metarhizium acridum.

Conidiation necessary for filamentous fungal survival and dispersal proceeds in two fashions, namely, normal conidiation through conidiophores differentiated from hyphae and microcycle conidiation through conidial budding. Normal conidiation has been well studied, whereas mechanisms underlying microcycle conidiation are still largely unknown. Here, we report that a gene (MaNsdD) homologous to NsdD in Aspergillus nidulans serves as a suppressor of normal conidiation but a positive regulator of hyphal development in Metarhizium acridum. Disruption of MaNsdD (ΔMaNsdD) resulted in microcycle conidiation and significantly descended in conidial resistance to heat while improved to UV irradiation. Transcriptomic analysis revealed that many genes involved in conidiation, cell division and cell wall formation were differentially expressed in ΔMaNsdD, and likely associated with the conidiation process. We found that a gene (MaAbaA) homologous to the core asexual development regulator AbaA in A. nidulans was negatively controlled by MaNsdD. Disruption of MaAbaA led to the abolition of the conidiation process of M. acridum. These findings unravel a novel regulatory mechanism of microcycle conidiation and add knowledge to the asexual conidiation pathway of filamentous fungi.

MaSln1, a Conserved Histidine Protein Kinase, Contributes to Conidiation Pattern Shift Independent of the MAPK Pathway in Metarhizium acridum.

As a conserved sensor kinase in the HOG-MAPK pathway, Sln1 plays distinct functions in different fungi. In this study, the roles of MaSln1 in Metarhizium acridum were analyzed using gene knockout and rescue strategies. Deletion of MaSln1 did not affect conidial germination, conidial yield, or resistance to chemical agents. However, fungal tolerance to heat shock and UV-B were significantly reduced after deletion of MaSln1. Insect bioassays showed that fungal pathogenicity was significantly impaired when MaSln1 was deleted. Further studies showed that MaSln1 did not affect either germination or appressorium formation of M. acridum on locust wings, but it significantly increased appressorium turgor pressure. In addition, disruption of MaSln1 resulted in a conidiation pattern shift in M. acridum. Microscopic observation revealed, however, that some genes located in the MAPK signaling pathway, including MaSho1, MaHog1, MaMk1, and MaSlt2, were not involved in the conidiation pattern shift on SYA medium (microcycle medium). Meanwhile, of the 143 differently expressed genes (DEGs) identified by RNA-seq, no genes related to the MAPK pathway were found, suggesting that MaSln1 regulation of the conidiation pattern shift was probably independent of the conserved MAPK signaling pathway. It was found that 22 of the 98 known DEGs regulated by MaSln1 were involved in mycelial growth, cell division, and cytoskeleton formation, indicating that MaSln1 likely regulates the expression of genes related to cell division and morphogenesis, thus regulating the conidiation pattern shift in M. acridum. IMPORTANCE The productivity and quality of conidia are both crucial for mycopesticides. In this study, we systematically analyzed the roles of MaSln1 in fungal pathogens. Most importantly, our results revealed that deletion of MaSln1 resulted in a conidiation pattern shift in M. acridum. However, some other genes, located in the MAPK signaling pathway, were not involved in the conidiation pattern shift. RNA-seq revealed no genes related to the MAPK pathway, suggesting that the regulation of the conidiation pattern shift by MaSln1 was probably independent of the conserved MAPK signaling pathway. This study provided a new insight into the functions of Sln1 and laid a foundation for exploring the mechanisms of conidiation pattern shifts in M. acridum.