Carbon catabolite repression (CCR) and de-repression (CCDR) are critical for fungal development and pathogenicity, yet the underlying regulatory mechanisms remain poorly understood in pathogenic fungi. Here, we identify a serine/threonine protein phosphatase catalytic subunit, Pp4c, as essential for growth, conidiation, virulence, and the utilization of carbohydrates and lipids in Magnaporthe oryzae. We demonstrate that the protein phosphatase 4 complex (Pp4c and Smek1 subunits), the AMP-activated protein kinase (AMPK) Snf1, and the transcriptional regulators CreA (repressor) and Crf1 (activator) collaboratively regulate the utilization of non-preferred carbon sources. Protein interaction and phosphorylation analyses reveal that under glucose-rich conditions, Snf1 and Smek1 directly regulate the phosphorylation status of CreA and Crf1. In contrast, under l-arabinose-rich conditions, Snf1 indirectly modulates the dephosphorylation of these transcription factors via Pp4c and Smek1. Phosphorylation-mediated activation or inactivation of CreA and Crf1 drives CCR and CCDR, thereby governing the metabolism of carbon sources derived from plant cell walls and contributing to fungal pathogenicity. These findings provide deep insights into the regulation of CCR and CCDR, emphasizing their significance in carbon metabolism and pathogenicity in phytopathogenic fungi. The protein phosphatase 4 complex, the Snf1 kinase, and the transcriptional regulators CreA and Crf1 collaboratively control carbon catabolite repression and de-repression in the rice blast fungus Magnaporthe oryzae.

In Magnaporthe oryzae, the Pmk1 MAP kinase signaling pathway regulates appressorium formation, plant penetration, effector secretion, and invasive growth. While the Mst11-Mst7-Pmk1 cascade was characterized two decades ago, knowledge of its signaling in the intracellular network remains limited. In this study, we demonstrate that the endosomal surface scaffolds Pmk1 MAPK signaling and Msb2 activates Ras2 on endosomes in M. oryzae. Protein colocalization demonstrated that Msb2, Ras2, Cap1, Mst50, Mst11, Mst7, and Pmk1 attach to late endosomal membranes. Damage to the endosome-vacuole transport system influences Pmk1 phosphorylation. When Msb2 senses a plant signal, it internalizes and activates Ras2 on endosome membrane surfaces, transmitting the signal to Pmk1 via Mst11 and Mst7. Signal-sensing and delivery proteins are ubiquitinated and sorted for degradation in late endosomes and vacuoles, terminating signaling. Plant penetration and lowered intracellular turgor are required for the transition from late endosomes to vacuoles in appressoria. Our findings uncover an effective mechanism that scaffolds and controls Pmk1 MAPK signaling through endosomal-vacuolar transport, offering new knowledge for the cytological and molecular mechanisms by which the Pmk1 MAPK pathway modulates development and pathogenicity in M. oryzae.

The cell wall is the first barrier against external adversity and plays roles in maintaining normal physiological functions of fungi. Previously, we reported a nucleosome assembly protein, MoNap1, in Magnaporthe oryzae that plays a role in cell wall integrity (CWI), stress response, and pathogenicity. Moreover, MoNap1 negatively regulates the expression of MoSMI1 encoded by MGG_03970. Here, we demonstrated that deletion of MoSMI1 resulted in a significant defect in appressorium function, CWI, cell morphology, and pathogenicity. Further investigation revealed that MoSmi1 interacted with MoOsm1 and MoMps1 and affected the phosphorylation levels of MoOsm1, MoMps1, and MoPmk1, suggesting that MoSmi1 regulates biological functions by mediating mitogen-activated protein kinase (MAPK) signalling pathway in M. oryzae. In addition, transcriptome data revealed that MoSmi1 regulates many infection-related processes in M. oryzae, such as membrane-related pathway and oxidation reduction process. In conclusion, our study demonstrated that MoSmi1 regulates CWI by mediating the MAPK pathway to affect development and pathogenicity of M. oryzae.

Rice blast is a serious threat to the safe production of grain crops such as rice and wheat. Sporulation, appressorium formation, and invasive growth of Magnaporthe oryzae are the key stages of the development and spread of rice blast epidemics. M. oryzae is a hemibiotrophic fungus that undergoes changes in available carbon sources during the infection cycle. Lipid is a major storage for M. oryzae spores and a major carbon source used in glycerol synthesis and turgor pressure generation in appressoria. The formation of a dense cell wall melanin layer is necessary for an appressorium to produce turgor and to be pathogenic. The plant cell wall is an important carbon source during the infection stage of M. oryzae . Transcription factors regulate gene expression in fungi and are key intermediates between the reception of external environmental signals and the control of development and pathogenicity in M. oryzae . The disease cycle of M. oryzae is controlled by some key transcription factors, such as sporulation by Cos1 and Hox2, appressorium formation by Sfl1, Hox7, and Vrf1, invasive growth by Mst12 and Mig1, and resistance to host basal immunity by Ap1 and Atf1. This review focuses on describing the key transcription factors of M. oryzae that regulate sporulation, appressorium formation, invasive growth, lipid metabolism, carbohydrate metabolism, melanin synthesis, oxidative response, and host basal immunity, as well as the working mechanism of the transcription factors.

Microhomology-mediated short tandem duplication (TD) is among specific mutational signatures associated with BRCA1 -deficient tumors. Several mechanisms have been proposed for its generation, but may not be applicable in repeat-less regions of the human genome. We thus developed a repeat-less TD reporter and a PCR-based site-specific TD assay to analyze short TDs induced by one-ended DNA double strand breaks (DSBs) converted from DNA nicks in Brca1 -deficient cells. We found that short TDs induced by DNA nicks are significantly stimulated in Brca1 -deficient cells. Analysis of TD products revealed that the TD formation is partly mediated by template switching of displaced nascent strand after allelic DNA synthesis. This suggests either allelic DNA synthesis or the strand annealing step of allelic break-induced replication might be more easily aborted in Brca1- deficient cells, thus promoting TD. Neither depletion of Rad51 or Brca2 nor inactivation of the Brca1 coiled-coil domain stimulated nick-induced TD, indicating that RAD51 loading by BRCA1 is dispensable for BRCA1-mediated TD suppression. These results together provide novel insights into the mechanisms underlying BRCA1 -linked TD formation in cancer.

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, beta-oxidation, and the glyoxylate cycle increased in the Delta smek1 mutant, which is consistent with Delta creA in which a carbon catabolite repressor CREA was deleted. In lipid medium, the expression of genes involved in long-chain fatty acid degradation, beta-oxidation, the glyoxylate cycle, and utilization of arabinose, ethanol, or glycerol decreased in the Delta smek1 mutant, which is consistent with Delta crf1 in which a transcription activator CRF1 required for carbon metabolism was deleted. Lipase activity, however, increased in the Delta smek1 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).

Paired SpCas9 nickases (SpCas9n) are an effective strategy to reduce off-target effect in genome editing. However, this approach is not efficient with 3'-overhanging ends, limiting its applications. In order to expand the utility of paired SpCas9n in genome editing, we tested the effect of the TREX2 3'-5' exonuclease on repair of 3'-overhanging ends. We found ectopic overexpression of Trex2 stimulates the efficiency of paired SpCas9n in genome disruption with 3'-overhanging ends up to 400-fold with little stimulation of off-target editing. TREX2 overexpressed preferentially deletes entire 3' overhangs but has no significant effect on 5' overhangs. Trex2 overexpression also stimulates genome disruption by paired SpCas9n that potentially generate short 3'-overhanging ends at overlapping SpCas9n target sites, suggesting sequential nicking of overlapping target sites by SpCas9n. This approach is further simplified with improved efficiency and safety by fusion of TREX2 and particularly its DNA-binding-deficient mutant to SpCas9n. Junction analysis at overlapping targets revealed the different extent of end resection of 3' single-stranded DNA (ssDNA) by free TREX2 and TREX2 fused to SpCas9n. SpCas9n-TREX2 fusion is more convenient and safer than overexpression of free TREX2 to process 3'-overhanging ends for efficient genome disruption by paired SpCas9n, allowing practical use of this TREX2-based strategy in genome editing.

In fungi, the methylcitrate cycle converts cytotoxic propionyl-coenzyme A (CoA) to pyruvate, which enters gluconeogenesis. The glyoxylate cycle converts acetyl-CoA to succinate, which enters gluconeogenesis. The tricarboxylic acid cycle is a central carbon metabolic pathway that connects the methylcitrate cycle, the glyoxylate cycle, and other metabolisms for lipids, carbohydrates, and amino acids. Fungal citrate synthase and 2-methylcitrate synthase as well as isocitrate lyase and 2-methylisocitrate lyase, each evolved from a common ancestral protein. Impairment of the methylcitrate cycle leads to the accumulation of toxic intermediates such as propionyl-CoA, 2-methylcitrate, and 2-methylisocitrate in fungal cells, which in turn inhibits the activity of many enzymes such as dehydrogenases and remodels cellular carbon metabolic processes. The methylcitrate cycle and the glyoxylate cycle synergistically regulate carbon source utilization as well as fungal growth, development, and pathogenic process in pathogenic fungi.

BACKGROUND:Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood. RESULTS:In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements. CONCLUSIONS:Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.

Magnaporthe oryzae infects rice, wheat and other grass crops through appressoria. The formation of the appressorium is regulated by the external environment, signal transduction pathways, and transcription factors. Transcription factors Vrf1 and Hox7 are involved in the regulation of appressorium formation. In this study, we demonstrate that Vrf1 and Hox7 play vital roles in coordinately regulating appressorium maturation. In strain 70-15, deletion of VRF1 resulted in the inability to continue melanization and maturation of the incipient appressorium, and deletion of HOX7 also resulted in defects in appressorium melanization and maturation. The defects in appressorium formation in Delta hox7 Delta vrf1 were similar to those in Delta hox7 and Delta vrf1. The gene expression profiles of the incipient appressoria at 5 h post-inoculation (hpi) showed that the expression levels of 704 genes (25.94 % of all differentially expressed genes in the three mutants) were significantly downregulated (606 genes) or upregulated (98 genes). In the appressoria of Delta hox7, Delta vrf1, and Delta hox7 Delta vrf1 at 5 hpi, the expression level of genes related to cell wall remodeling was changed. Genes for melanin synthesis, chitin and glucan degradation, and extracellular cell wall degrading enzyme were significantly downregulated, while genes for chitin and glucan synthesis were upregulated. After 8 hpi, the incipient appressoria of Delta hox7, Delta vrf1, and Delta hox7 Delta vrf1 regerminated and formed swollen hyphal-like structures with multiple nuclei. The ratio of the nuclear number of the hyphal-like structures of Delta hox7, Delta hox7 Delta vrf1, and Delta vrf1 was close to 6:4:2 at 24 hpi. Therefore, although Vrf1 and Hox7 are somewhat functionally different, they synergistically regulate appressorium maturation in M. oryzae.

Analysis of human cancer genome sequences has revealed specific mutational signatures associated with BRCA1 -deficient tumors, but the underlying mechanisms remain poorly understood. Here, we show that one-ended DNA double strand breaks (DSBs) converted from CRISPR/Cas9-induced nicks by DNA replication, not two-ended DSBs, cause more characteristic chromosomal aberrations and micronuclei in Brca1 -deficient cells than in wild-type cells. BRCA1 is required for efficient homologous recombination of these nick-converted DSBs and suppresses bias towards long tract gene conversion and tandem duplication (TD) mediated by two-round strand invasion in a replication strand asymmetry. However, aberrant repair of these nick-converted one-ended DSBs, not that of two-ended DSBs in Brca1 -deficient cells, generates mutational signatures such as small indels with microhomology (MH) at the junctions, translocations and small MH-mediated TDs, resembling those in BRCA1 -deficient tumors. These results suggest a major contribution of DNA nicks to mutational signatures associated with BRCA1 deficiency in cancer and the underlying mechanisms.

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 appressorium is a specialized structure that is differentiated from Magnaporthe oryzae spores that can infect host cells. In the process of cellular transformation from spore to appressorium, the contents inside the spores are transferred into appressoria, accompanied by major differences in the gene expression model. In this study, we reported a transcription factor (TF), Pcf1, which was depressed at the transcription level and degraded at the protein level in nuclei of incipient appressoria at four hpi (hours post inoculation). To investigate its degradation mechanism, the interacting proteins of Pcf1 were identified using an immunoprecipitation-mass spectrometry (IP-MS) assay. Yeast two-hybrid (Y2H) and co-IP (co-immunoprecipitation) assays confirmed that Pcf1 interacted with the casein kinase 2 (CK2) holoenzyme through direct combination with the CKb2 subunit. Moreover, Pcf1 was ubiquitinated in the hyphae. These changes in Pcf1 protein levels in nuclei provide a new clue of how TFs are degraded during appressorium formation: temporarily unnecessary TFs in spores are phosphorylated through interacting with CK2 enzyme and are then ubiquitinated and digested by the ubiquitin proteasome system (UPS).

Magnaporthe oryzae is the causal agent of rice blast, leading to significant reductions in rice and wheat productivity. Nap1 is a conserved protein in eukaryotes involved in diverse physiological processes, such as nucleosome assembly, histone shuttling between the nucleus and cytoplasm, transcriptional regulation, and the cell cycle. Here, we identified Nap1 and characterized its roles in fungal development and virulence in M. oryzae. MoNap1 is involved in aerial hyphal and conidiophore differentiation, sporulation, appressorium formation, plant penetration, and virulence. ΔMonap1 generated a small, elongated, and malformed appressorium with an abnormally organized septin ring on hydrophobic surfaces. ΔMonap1 was more sensitive to cell wall integrity stresses but more resistant to microtubule stresses. MoNap1 interacted with histones H2A and H2B and the B-type cyclin (Cyc1). Moreover, a nuclear export signal (NES) domain is necessary for Nap1’s roles in the regulation of the growth and pathogenicity of M. oryzae. In summary, NAP1 is essential for the growth, appressorium formation, and pathogenicity of M. oryzae.

Rice straw was substituted for sawdust at five different ratios of 0, 20%, 40%, 60%, and 80% (Control, RS20, RS40, RS60 and RS80, respectively) to obtain five kinds of Lentinula edodes . The effects of adding cropped rice straw to substrate formulas on the proximate composition and non-volatile taste compounds in mushrooms were investigated. The control group had the highest level of MY and BE among the five formulations. The protein levels in mushrooms decreased with the addition of rice straw and the ash levels increased. We found that trehalose, mannitol, and arabitol were the main soluble sugars in the five kinds of mushrooms. The contents of total free amino acids varied from 16.29 to 24.59 mg/g and the highest level of free amino acids was found in mushrooms cultivated from RS20 and RS40. Moreover, the addition of rice straw improved the contents of monosodium glutamate (MSG)-like amino acids in mushrooms. The 5′-Nucleotide levels ranged from 1.66 to 4.48 mg/g and equivalent umami concentration (EUC) value increased with the addition of rice straw. Our results suggest that rice straw is a potential substitute for sawdust to cultivate L. edodes with more non-volatile taste compounds.

Endogenous formaldehyde is generated as a normal metabolite via bio-catalysis of γ-glutamyl transpeptidase (GGT) and L-cysteine sulfoxide lyase (C-S lyase) during the growth and development of Lentinula edodes. In this study, we investigated the mRNA and protein expression levels, the activities of GGT and C-S lyase, and the endogenous formaldehyde content in L. edodes at different growth stages. With the growth of L. edodes, a decrease was found in the mRNA and protein expression levels of GGT, while an increase was observed in the mRNA and protein expression levels of C-S lyase as well as the activities of GGT and C-S lyase. Our results revealed for the first time a positive relationship of formaldehyde content with the expression levels of Csl (encoding Lecsl) and Lecsl (C-S lyase protein of Lentinula edodes) as well as the enzyme activities of C-S lyase and GGT during the growth of L. edodes. This research provided a molecular basis for understanding and controlling the endogenous formaldehyde formation in Lentinula edodes in the process of growth.

Volatile organosulfur compounds are the main components that contribute to the unique aroma of dried Lentinula edodes. They are mainly generated during the hot-air drying process, and cysteine desulfurase is the key enzyme in this process. Temperature may be an essential factor of volatile organosulfur compound production by influencing the expression of the cysteine desulfurase gene. In this study, the promoter sequence of the cysteine desulfurase gene (pCS) was cloned and analyzed using bioinformatics tools. A series of 5′deletion fragments and site-directed mutations of pCS were constructed to identify the element that responds to heat stress. Six heat shock transcription factor (HSTF) binding sites were predicted by SCPD (The Promoter Database of Saccharomyces cerevisiae) and three of the binding sites were predicted by Yeastract (Yeast Search for Transcriptional Regulators and Consensus Tracking) in pCS. The results indicated that pCS was able to drive the expression of the EGFP (Enhanced Green Fluorescent Protein) gene in L. edodes. Moreover, the fluorescence intensity increased after heat stress. The changes in fluorescence intensity of different 5′deletion fragments showed that the heat response region was located between −500 bp and −400 bp in pCS. The site-directed mutation analysis further showed that the heat-inducible element was between −490 bp and −500 bp (TTTCTAGAAT) in pCS. Our results provide molecular insight for studying the formation of volatile organosulfur compounds in dried L. edodes.

Volatile sulfur compounds gradually develop in Lentinula edodes after hot-air drying, and many genes are involved in the generation of these sulfur compounds. The expression stability of reference genes may vary in a particular experimental treatment when analyzing their expressions by quantitative real-time polymerase chain reaction (qRT-PCR). In this study, the expression profile of 17 candidate genes was assessed in L. edodes under treatment at 50 °C for 0, 1, 2, and 3 h, and the expression stability of each reference gene was analyzed by three statistical algorithms, including geNorm, NormFinder, and BestKeeper. Results indicated that the two optimal reference genes for mycelium and fruiting body were CAC and DAHP as well as CAC and NUP, respectively. Additionally, CAC and DAHP were found to be the two most stable reference genes across the mycelium and fruiting body set. Our results will provide a genetic foundation for further research on the metabolism genes of sulfur compounds in L. edodes.

DnaJ proteins, termed heat shock proteins based on their molecular weight, function as molecular chaperones that play critical roles in regulating organism growth and development as well as adaptation to the environment. However, little has been reported on their gene function in higher basidiomycetes. Here, the heat shock protein 40 (LeDnaJ) gene was cloned and characterized from Lentinula edodes. RNA interference was used to explore the function of LeDnaJ in response to heat stress and Trichoderma atroviride. Integration of the target gene into the L. edodes genome was confirmed by Southern blot analysis, and the silence efficiency of LeDnaJ was analyzed by qRT-PCR. The results revealed that LeDnaJ silence caused defects in mycelial growth and resistance to heat stress and T. atroviride, but increased the mycelial density compared with the wild type (WT) strain S606. Additionally, the IAA content showed a more than 10-fold increase in the WT after heat stress, but an about two-fold increase in the two LeDnaJ RNAi transfortants (LeDnaJ-i-6 and LeDnaJ-i-8). Previous study has shown that enhanced IAA (indole-3-acetic acid) content enhanced the thermotolerance of the heat-sensitive strain YS3357. In this study, it was documented that IAA amendments could partly restore the resistance to T. atroviride and thermotolerance of the two LeDnaJ RNAi transformants. Overall, LeDnaJ is nvolved in fungal growth, T. atroviride resistance, and thermotolerance by regulating the IAA biosynthesis in L. edodes.