Ustilaginoidea virens, an emerging pathogen of rice: the dynamic interplay between the pathogen virulence strategies and host defense.

MAIN CONCLUSION: The Ustilaginoidea virens -rice pathosystem has been used as a model for flower-infecting fungal pathogens. The molecular biology of the interactions between U. virens and rice, with an emphasis on the attempt to get a deeper comprehension of the false smut fungus's genomes, proteome, host range, and pathogen biology, has been investigated. Meta-QTL analysis was performed to identify potential QTL hotspots for use in marker-assisted breeding. The Rice False Smut (RFS) caused by the fungus Ustilaginoidea virens currently threatens rice cultivators across the globe. RFS infects rice panicles, causing a significant reduction in grain yield. U. virens can also parasitize other hosts though they play only a minor role in its life cycle. Furthermore, because it produces mycotoxins in edible rice grains, it puts both humans and animals at risk of health problems. Although fungicides are used to control the disease, some fungicides have enabled the pathogen to develop resistance, making its management challenging. Several QTLs have been reported but stable gene(s) that confer RFS resistance have not been discovered yet. This review offers a comprehensive overview of the pathogen, its virulence mechanisms, the genome and proteome of U. virens, and its molecular interactions with rice. In addition, information has been compiled on reported resistance QTLs, facilitating the development of a consensus genetic map using meta-QTL analysis for identifying potential QTL hotspots. Finally, this review highlights current developments and trends in U. virens-rice pathosystem research while identifying opportunities for future investigations.

RNA silencing is a key regulatory mechanism in the biocontrol fungus Clonostachys rosea-wheat interactions.

BACKGROUND: Small RNA (sRNAs)- mediated RNA silencing is emerging as a key player in host-microbe interactions. However, its role in fungus-plant interactions relevant to biocontrol of plant diseases is yet to be explored. This study aimed to investigate Dicer (DCL)-mediated endogenous and cross-kingdom gene expression regulation in the biocontrol fungus Clonostachys rosea and wheat roots during interactions. RESULTS: C. rosea Δdcl2 strain exhibited significantly higher root colonization than the WT, whereas no significant differences were observed for Δdcl1 strains. Dual RNA-seq revealed the upregulation of CAZymes, membrane transporters, and effector coding genes in C. rosea, whereas wheat roots responded with the upregulation of stress-related genes and the downregulation of growth-related genes. The expression of many of these genes was downregulated in wheat during the interaction with DCL deletion strains, underscoring the influence of fungal DCL genes on wheat defense response. sRNA sequencing identified 18 wheat miRNAs responsive to C. rosea, and three were predicted to target the C. rosea polyketide synthase gene pks29. Two of these miRNAs (mir_17532_x1 and mir_12061_x13) were observed to enter C. rosea from wheat roots with fluorescence analyses and to downregulate the expression of pks29, showing plausible cross-kingdom RNA silencing of the C. rosea gene by wheat miRNAs. CONCLUSIONS: We provide insights into the mechanisms underlying the interaction between biocontrol fungi and plant roots. Moreover, the study sheds light on the role of sRNA-mediated gene expression regulation in C. rosea-wheat interactions and provides preliminary evidence of cross-kingdom RNA silencing between plants and biocontrol fungi.

Enhanced heterologous gene expression in Trichoderma reesei by promoting multicopy integration.

Trichoderma reesei displays a high capability to produce extracellular proteins and therefore is used as a platform for the expression of heterologous genes. In a previous study, an expression cassette with the constitutive tef1 promoter and the cbh1 terminator compatible with flow cytometry analysis was developed. Independent transformants obtained by a random integration into the genome of a circular plasmid containing the expression cassette showed a wide range of fluorescence levels. Whole genome sequencing was conducted on eight of the transformed strains using two next-generation sequencing (NGS) platforms: Illumina paired-end sequencing and Oxford Nanopore. In all strains, the expression plasmid was inserted at the same position in the genome, i.e., upstream of the tef1 gene, indicating an integration by homologous recombination. The different levels of fluorescence observed correspond to different copy numbers of the plasmid. Overall, the integration of a circular plasmid with the green fluorescence protein (egfp) transgene under the control of tef1 promoter favors multicopy integration and allows over-production of this heterologous protein on glucose. In conclusion, an expression system based on using the tef1 promotor could be one of the building blocks for improving high-value heterologous protein production by increasing the copy number of the encoding genes into the genome of the platform strain. KEY POINTS: • Varied eGFP levels from tef1 promoter and cbh1 terminator expression. • Whole genome sequencing on short and long reads platforms reveals various plasmid copy numbers in strains. • Plasmids integrate at the same genomic site by homologous recombination in all strains.

Integration of Transcriptomics and WGCNA to Characterize Trichoderma harzianum-Induced Systemic Resistance in Astragalus mongholicus for Defense against Fusarium solani.

Beneficial fungi of the genus Trichoderma are among the most widespread biocontrol agents that induce a plant's defense response against pathogens. Fusarium solani is one of the main pathogens that can negatively affect Astragalus mongholicus production and quality. To investigate the impact of Trichoderma harzianum on Astragalus mongholicus defense responses to Fusarium solani, A. mongholicus roots under T. harzianum + F. solani (T + F) treatment and F. solani (F) treatment were sampled and subjected to transcriptomic analysis. A differential expression analysis revealed that 6361 differentially expressed genes (DEGs) responded to T. harzianum induction. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the 6361 DEGs revealed that the genes significantly clustered into resistance-related pathways, such as the plant-pathogen interaction pathway, phenylpropanoid biosynthesis pathway, flavonoid biosynthesis pathway, isoflavonoid biosynthesis pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and plant hormone signal transduction pathway. Pathway analysis revealed that the PR1, formononetin biosynthesis, biochanin A biosynthesis, and CHIB, ROS production, and HSP90 may be upregulated by T. harzianum and play important roles in disease resistance. Our study further revealed that the H2O2 content was significantly increased by T. harzianum induction. Formononetin and biochanin A had the potential to suppress F. solani. Weighted gene coexpression network analysis (WGCNA) revealed one module, including 58 DEGs associated with T. harzianum induction. One core hub gene, RPS25, was found to be upregulated by T. harzianum, SA (salicylic acid) and ETH (ethephon). Overall, our data indicate that T. harzianum can induce induced systemic resistance (ISR) and systemic acquired resistance (SAR) in A. mongholicus. The results of this study lay a foundation for a further understanding of the molecular mechanism by which T. harzianum induces resistance in A. mongholicus.

Growth promotion and modulation of the soybean microbiome INTACTA RR PRO with the application of the fungi Trichoderma harzianum and Purpureocillum lilacinum.

Soybean is an economically important crop for animal and human nutrition. Currently, there is a lack of information on the effects of Trichoderma harzianum and Purpureocillum lilacinum on INTACTA RR PRO transgenic soybean plants. The present study evaluated the application of T. harzianum and P. lilacinum under field conditions. The results revealed a significant increase in soybean yield at 423 kg ha-1 in response to the application of P. lilacinum compared with the control treatment. In addition, the application of P. lilacinum promoted a significant increase in phosphorus levels in the plant leaves, and there were significant correlations between the increase in taxon abundance for the genus Erwinia and productivity and the average phosphorus and nitrogen content for the plant leaves, for the taxon Bacillus and nitrogen content and productivity, and for the taxon Sphingomonas and nitrogen content. The Bradyrhizobium taxon was identified in the P. lilacinum treatment as a taxon linking two different networks of taxa and is an important taxon in the microbiota. The results show that the application of the fungus P. lilacinum can increase the productivity of soybean INTACTA RR PRO and that this increase in productivity may be a function of the modulation of the microbiota composition of the plant leaves by the P. lilacinum effect.

A secreted fungal laccase targets the receptor kinase OsSRF3 to inhibit OsBAK1-OsSRF3-mediated immunity in rice.

The identification effector targets and characterization of their functions are crucial for understanding pathogen infection mechanisms and components of plant immunity. Here, we identify the effector UgsL, a ustilaginoidin synthetase with a key role in regulating virulence of the rice false smut fungus Ustilaginoidea virens. Heterologous expression of UgsL in rice (Oryza sativa) enhances plant susceptibility to multiple pathogens, and host-induced gene silencing of UgsL enhances plant resistance to U. virens, indicating that UgsL inhibits rice immunity. UgsL interacts with STRUBBELIG RECEPTOR KINASE 3 (OsSRF3). Genome editing and overexpression of OsSRF3 demonstrate that OsSRF3 plays a pivotal role in the resistance of rice to multiple pathogens. Remarkably, overexpressing OsSRF3 enhances resistance without adversely affecting plant growth or yield. We show that BRASSINOSTEROID RECEPTOR-ASSOCIATED KINASE 1 (OsBAK1) interacts with and phosphorylates OsSRF3 to activate pathogen-triggered immunity, inducing the mitogen-activated protein kinase cascade, a reactive oxygen species burst, callose deposition, and expression of defense-related genes. UgsL interferes with the phosphorylation of OsSRF3 by OsBAK1. Furthermore, UgsL mediates OsSRF3 degradation by facilitating its association with the ubiquitin-26S proteasome. Our results reveal that OsSRF3 positively regulates immunity in rice and that UgsL mediates its degradation, thereby inhibiting the activation of OsBAK1-OsSRF3-mediated immune pathways.

Harnessing Lecanicillium attenuatum: A novel strategy for combatting Nilaparvata lugens in rice fields.

Nilaparvata lugens is a notorious rice pest causing significant annual yield and economic losses. The use of entomopathogenic fungi offers a promising and eco-friendly approach to sustainable pest management programs. However, research in this area is currently limited to a few specific types of insects and other arthropods. This study aimed to analyze the biocontrol potential of Lecanicillium attenuatum against N. lugens. Bioassays showed that L. attenuatum 3166 induced >80% mortality in N. lugens following 7 d exposure. Greenhouse and field investigations demonstrated that L. attenuatum 3166 application leads to a substantial reduction in N. lugens populations. Under greenhouse conditions, fluorescence was detected in GFP-labeled L. attenuatum 3166 hyphae enveloping the bodies of N. lugens. In field trials, L. attenuatum 3166 treatment exhibited a control efficacy of up to 68.94% at 14 d post-application, which was comparable to that of the commercial entomopathogenic fungal agent. Genomic sequencing of L. attenuatum 3166 revealed a comprehensive array of genes implicated in its infestation and lethality. Further, the transcriptome sequencing analysis highlighted the elevated expression levels of genes encoding proteases, chitinases, cutinases, and phospholipases. Our findings highlight the potential of L. attenuatum 3166 as an effective biological control agent against N. lugens.

Characteristics of a Novel Zearalenone Lactone Hydrolase ZHRnZ and Its Thermostability Modification.

Zearalenone (ZEN) is a toxic secondary metabolite produced by the Fusarium fungi, which widely contaminates grains, food, and feed, causing health hazards for humans and animals. Therefore, it is essential to find effective ZEN detoxification methods. Enzymatic degradation of ZEN is believed to be an eco-friendly detoxification strategy, specifically thermostable ZEN degradation enzymes are needed in the food and feed industry. In this study, a novel ZEN lactone hydrolase ZHRnZ from Rosellinia necatrix was discovered using bioinformatic and molecular docking technology. The recombinant ZHRnZ showed the best activity at pH 9.0 and 45 °C with more than 90% degradation for ZEN, α-zearalenol (α-ZOL), ß-zearalenol (ß-ZOL) and α-zearalanol (α-ZAL) after incubation for 15 min. We obtained 10 mutants with improved thermostability by single point mutation technology. Among them, mutants E122Q and E122R showed the best performance, which retained more than 30% of their initial activity at 50 °C for 2 min, and approximately 10% of their initial activity at 60 °C for 1 min. The enzymatic kinetic study showed that the catalytic efficiency of E122R was 1.3 times higher than that of the wild-type (WT). Comprehensive consideration suggests that mutant E122R is a promising hydrolase to detoxify ZEN in food and feed.

Neurotransmitter acetylcholine mediates the mummification of Ophiocordyceps sinensis-infected Thitarodes xiaojinensis larvae.

Parasites can manipulate host behavior to facilitate parasite transmission. One such host-pathogen interaction occurs between the fungus Ophiocordyceps sinensis and the ghost moth Thitarodes xiaojinensis. O. sinensis is involved in the mummification process of infected host larvae. However, the underlying molecular and chemical mechanism for this phenomenon is unknown. We characterized the small molecules regulating host behaviors and the altered metabolites in infected and mummified host larvae. Lipid-related metabolites, such as phosphatidylcholine, were identified in infected and mummified larvae. Decreased levels of the neurotransmitter acetylcholine (ACh) and elevated choline levels were observed in the brains of both the infected and mummified larvae. The aberrant activity of acetylcholinesterase (AChE) and relative mRNA expression of ACE2 (acetylcholinesterase) may mediate the altered transformation between ACh and choline, leading to the brain dysfunction of mummified larvae. Caspofungin treatment inhibited the mummification of infected larvae and the activity of AChE. These findings indicate the importance of ACh in the mummification of host larvae after O. sinensis infection.IMPORTANCEOphiocordyceps sinensis-infected ghost moth larvae are manipulated to move to the soil surface with their heads up in death. A fruiting body then grows from the caterpillar's head, eventually producing conidia for dispersal. However, the underlying molecular and chemical mechanism has not been characterized. In this study, we describe the metabolic profile of Thitarodes xiaojinensis host larvae after O. sinensis infection. Altered metabolites, particularly lipid-related metabolites, were identified in infected and mummified larvae, suggesting that lipids are important in O. sinensis-mediated behavioral manipulation of host larvae. Decreased levels of the neurotransmitter acetylcholine were observed in both infected and mummified larvae brains. This suggests that altered or reduced acetylcholine can mediate brain dysfunction and lead to aberrant behavior. These results reveal the critical role of acetylcholine in the mummification process of infected host larvae.