First telomere-to-telomere gapless assembly of the rice blast fungus Pyricularia oryzae.

Rice blast caused by Pyricularia oryzae (syn., Magnaporthe oryzae) was one of the most destructive diseases of rice throughout the world. Genome assembly was fundamental to genetic variation identification and critically impacted the understanding of its ability to overcome host resistance. Here, we report a gapless genome assembly of rice blast fungus P. oryzae strain P131 using PacBio, Illumina and high throughput chromatin conformation capture (Hi-C) sequencing data. This assembly contained seven complete chromosomes (43,237,743 bp) and a circular mitochondrial genome (34,866 bp). Approximately 14.31% of this assembly carried repeat sequences, significantly greater than its previous assembled version. This assembly had a 99.9% complement in BUSCO evaluation. A total of 14,982 genes protein-coding genes were predicted. In summary, we assembled the first telomere-to-telomere gapless genome of P. oryzae, which would be a valuable genome resource for future research on the genome evolution and host adaptation.

Plant immunity suppression by an exo-ß-1,3-glucanase and an elongation factor 1α of the rice blast fungus.

Fungal cell walls undergo continual remodeling that generates ß-1,3-glucan fragments as products of endo-glycosyl hydrolases (GHs), which can be recognized as pathogen-associated molecular patterns (PAMPs) and trigger plant immune responses. How fungal pathogens suppress those responses is often poorly understood. Here, we study mechanisms underlying the suppression of ß-1,3-glucan-triggered plant immunity by the blast fungus Magnaporthe oryzae. We show that an exo-ß-1,3-glucanase of the GH17 family, named Ebg1, is important for fungal cell wall integrity and virulence of M. oryzae. Ebg1 can hydrolyze ß-1,3-glucan and laminarin into glucose, thus suppressing ß-1,3-glucan-triggered plant immunity. However, in addition, Ebg1 seems to act as a PAMP, independent of its hydrolase activity. This Ebg1-induced immunity appears to be dampened by the secretion of an elongation factor 1 alpha protein (EF1α), which interacts and co-localizes with Ebg1 in the apoplast. Future work is needed to understand the mechanisms behind Ebg1-induced immunity and its suppression by EF1α.

The rice blast fungus SR protein 1 regulates alternative splicing with unique mechanisms.

Serine/arginine-rich (SR) proteins are well known as splicing factors in humans, model animals and plants. However, they are largely unknown in regulating pre-mRNA splicing of filamentous fungi. Here we report that the SR protein MoSrp1 enhances and suppresses alternative splicing in a model fungal plant pathogen Magnaporthe oryzae. Deletion of MoSRP1 caused multiple defects, including reduced virulence and thousands of aberrant alternative splicing events in mycelia, most of which were suppressed or enhanced intron splicing. A GUAG consensus bound by MoSrp1 was identified in more than 94% of the intron or/and proximate exons having the aberrant splicing. The dual functions of regulating alternative splicing of MoSrp1 were exemplified in enhancing and suppressing the consensus-mediated efficient splicing of the introns in MoATF1 and MoMTP1, respectively, which both were important for mycelial growth, conidiation, and virulence. Interestingly, MoSrp1 had a conserved sumoylation site that was essential to nuclear localization and enhancing GUAG binding. Further, we showed that MoSrp1 interacted with a splicing factor and two components of the exon-joining complex via its N-terminal RNA recognition domain, which was required to regulate mycelial growth, development and virulence. In contrast, the C-terminus was important only for virulence and stress responses but not for mycelial growth and development. In addition, only orthologues from Pezizomycotina species could completely rescue defects of the deletion mutants. This study reveals that the fungal conserved SR protein Srp1 regulates alternative splicing in a unique manner.

First Report of Leaf Spot on Stephania tetrandra Caused by Albifimbria verrucaria in China.

Stephania tetrandra S. Moore belongs to the family Menispermaceae and is a Chinese medicinal plant widely distributed in tropical and subtropical regions of Asia and Africa. The root can be used for a variety of treatments (Jiang et al. 2020). In August 2021, leaf spot symptoms were observed on S. tetrandra cultivated in Jiangxi (114.456E, 27.379N, southern China). The disease symptoms included a slight constriction of the leaves, with irregularly shaped brown to black spots with well-defined borders. Severely affected leaves were shed by the plant. In order to determine the cause, symptomatic leaves were surface-disinfested with 0.6% NaOCl for 2 min, and rinsed twice in sterile water, then incubated on moist paper towels at 26°C in the dark for 2 days. Cream-colored sporodochia were observed within the leaf spots, turning dark green to black within 16 hours. A slow-growing white fungus was isolated from 95% of the samples (n = 30) on PDA. Dark green sporodochia emerged after 7 to 10 days of incubation, and released tip-end oval, non-septate, hyaline conidia measuring 6.7 to 8.5 µm (mean 7.5 µm, n = 50) by 2.0 to 3.3 µm (mean 2.7 µm, n = 50). Concentric rings were interspersed with sporodochia on the continually incubated mycelium. The morphological characteristics of the isolates matched the description of Albifimbria (Lombard et al. 2016). Nucleotide sequences, amplified from isolate FJL5C using primers of the internal transcribed spacer (ITS) (White et al. 1990), calmodulin (cmdA; Carbone and Kohn 1999), and RNA polymerase II second largest subunit (rpb2; O'Donnell et al. 2007), were deposited in GenBank under accession numbers OM317911, OM386815, and OM386816. A BLASTn analysis of the sequences showed 100% identity with the type strain CBS 328.52 (Lombard et al. 2016) of Albifimbria verrucaria (syn. Myrothecium verrucaria) for ITS, and 99% for cmdA and rpb2 (KU845893, KU845875, and KU845931, respectively). A phylogenetic tree generated using the three sequences showed that the isolate from S. tetrandra grouped with the A. verrucaria isolates, but away from other species of Albifimbria. These results together with the lack of a pale luteus exudate produced by A. viridis (Lombard et al. 2016) implied that the isolate was A. verrucaria. The culture was deposited in Guangdong Microbial Culture Collection Center (GDMCC 3.716). To verify pathogenicity, conidial suspension (106 conidia/mL in 0.05% Tween 20 solution) was sprayed onto six healthy plants. Six other plants sprayed with the Tween 20 solution alone served as controls. All plants were incubated in the dark at 26°C and 95% humidity for 30 hours, then transferred to a greenhouse at 26°C and 12 hours of illumination per day for 2 to 3 days. Inoculated leaves developed similar symptoms to those described above, whereas control plants remained healthy. The same pathogen was isolated from the diseased leaves, with the same morphological and molecular traits as those from the field plants. This experiment fulfilled Koch's postulates and confirmed that A. verrucaria causes leaf spots on S. tetrandra. This pathogen has been reported to cause disease in a wide range of weeds, legumes, and crop plants (Herman et al. 2020). To our knowledge, this is the first report of A. verrucaria causing leaf spots on S. tetrandra in natural or controlled environments. The disease can seriously threaten S. tetrandra on growth and yield loss.

First Report of Sclerotium rolfsii Causing Southern Blight on Stephania tetrandra in China.

Stephania tetrandra S. Moore is a perennial liana and is widely cultivated in southern China for traditional Chinese medicine as a diuretic, anti-inflammatory, and antirheumatic treatment (Jiang et al. 2020). In August 2021, it was observed that a severe stem rot disease affected St. tetrandra cultivated in Anfu, Jiangxi province, China (114°27'26" E, 27°22'46" N). The disease symptoms included constriction and rot at the base of the stem, and covered with a layer of white mycelia. The plants above-ground finally wilted and dried with a disease incidence ranging from 8% to 16%. Lots of dried plants formed withered patches of field. Sections (1.0~2.0 cm) from browning stem tissues were surface-disinfected with 75% ethanol for 15 s, followed by 60 s in 4% NaClO, rinsed twice in sterile water, dried on sterilized filter paper, placed on potato dextrose agar (PDA), and incubated at 26°C in the dark for 3 days. A white rhizomorphic fungal mycelium, that is similar to the mycelium of strain FJSR0 on the surface of an infected plant in the field, was isolated from the cultured tissues with 67% frequency. When incubated on PDA, white and fluffy mycelia with even margins and a slight halo formed. Mycelia-produced clamp connections were observed. Colonies grew quickly and covered the dish (diameter: 9 cm) in 5 or 6 days. After that, sclerotia were initially white, then turned yellow, and chestnut brown at maturity. Spherical and subspherical sclerotia were observed after 8 days, with each plate containing 448 to 634 sclerotia (0.8 to 1.4 mm diameter; mean = 0.94 mm; n = 50). On the basis of morphology, the pathogen was similar to Sclerotium rolfsii Sacc. [teleomorph: Athelia rolfsii (Curzi) Tu & Kimbrough] (Sun et al. 2020; Ling et al. 2021). For molecular confirmation, the internal transcribed spacer (ITS) region with approximately 680 bp was amplified from strains FJRS0 and FJRS1 using primers ITS1/ITS4 (White et al. 1990). Two distinct types (different in one SNP and one 1-bp InDel) of ITS sequences were obtained from each isolate, and all isolates contain the two types (FJSR0: ON972516, ON972517; FJSR1: ON972520, ON972518). BLAST analysis of each type found that the hits, with identities >99%, are A. rolfsii except for two Sc. delphinii sequences (GU567775.1 and MK073010.1). Phylogenetic analysis placed strains FJSR0 and FJSR1 in the same clade as Sc. rolfsii but away from Sc. delphinii based on the previous method (Sun et al. 2021). Both morphological and molecular characteristics confirmed that the strains were Sc. rolfsii. For pathogenicity tests, PDA plugs (8 mm in diameter) covered with 5-day-old fungal mycelium were inoculated at the stem bases of three healthy St. tetrandra seedings and incubated at 26℃ and relative humidity of 80%. On the fifth day, inoculated plants were wilting. The infected stem bases turned brown to black and constricted as previously observed in the field. Some leaves, infected by the mycelium expanded from the PDA plugs, developed an orange and irregular spot. Sclerotia were observed 20 days post inoculation. In contrast, the leaves and stems of non-inoculated control plants remained symptomless. Pathogenicity tests were repeated three times. The fungus was reisolated consistently from each symptomatic tissue, thus completing Koch's postulates. Although Sc. rolfsii has been previously reported to cause a southern blight symptoms on vegetables, ornamentals, grass, and medicinal and leguminous crops (Sun et al. 2020; Ling et al. 2021), this is the first report of Sc. rolfsii causing similar symptoms of southern blight on St. tetrandra in China.

The Rice Malectin Regulates Plant Cell Death and Disease Resistance by Participating in Glycoprotein Quality Control.

In animals, malectin is well known to play an essential role in endoplasmic reticulum quality control (ERQC) by interacting with ribophorin I, one unit of the oligosaccharyltransferase (OST) complex. However, the functions of malectin in plants remain largely unknown. Here, we demonstrate the rice OsMLD1 is an ER- and Golgi-associated malectin protein and physically interacts with rice homolog of ribophorin I (OsRpn1), and its disruption leads to spontaneous lesion mimic lesions, enhanced disease resistance, and prolonged ER stress. In addition, there are many more N-glycosites and N-glycoproteins identified from the mld1 mutant than wildtype. Furthermore, OsSERK1 and OsSERK2, which have more N-glycosites in mld1, were demonstrated to interact with OsMLD1. OsMLD1 can suppress OsSERK1- or OsSERK2-induced cell death. Thus, OsMLD1 may play a similar role to its mammalian homologs in glycoprotein quality control, thereby regulating cell death and immunity of rice, which uncovers the function of malectin in plants.

Comparative Secretome Analysis of Magnaporthe oryzae Identified Proteins Involved in Virulence and Cell Wall Integrity.

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant-fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.

Prp19-associated splicing factor Cwf15 regulates fungal virulence and development in the rice blast fungus.

The splicing factor Cwf15 is an essential component of the Prp19-associated component of the spliceosome and regulates intron splicing in several model species, including yeasts and human cells. However, the roles of Cwf15 remain unexplored in plant pathogenic fungi. Here, we report that MoCWF15 in the rice blast fungus Magnaporthe oryzae is non-essential to viability and important to fungal virulence, growth and conidiation. MoCwf15 contains a putative nuclear localization signal (NLS) and is localized into the nucleus. The NLS sequence but not the predicted phosphorylation site or two sumoylation sites was essential for the biological functions of MoCwf15. Importantly, MoCwf15 physically interacted with the Prp19-associated splicing factors MoCwf4, MoSsa1 and MoCyp1, and negatively regulated protein accumulations of MoCyp1 and MoCwf4. Furthermore, with the deletion of MoCWF15, aberrant intron splicing occurred in near 400 genes, 20 of which were important to the fungal development and virulence. Taken together, MoCWF15 regulates fungal growth and infection-related development by modulating the intron splicing efficiency of a subset of genes in the rice blast fungus.

A novel glycine-rich domain protein, GRDP1, functions as a critical feedback regulator for controlling cell death and disease resistance in rice.

Lesion mimic mutants constitute a valuable genetic resource for unraveling the signaling pathways and molecular mechanisms governing the programmed cell death and defense responses of plants. Here, we identified a lesion mimic mutant, spl-D, from T-DNA insertion rice lines. The mutant exhibited higher accumulation of H2O2, spontaneous cell death, decreased chlorophyll content, up-regulation of defense-related genes, and enhanced disease resistance. The causative gene, OsGRDP1, encodes a cytosol- and membrane-associated glycine-rich domain protein. OsGRDP1 was expressed constitutively in all of the organs of the wild-type plant, but was up-regulated throughout plant development in the spl-D mutant. Both the overexpression and knockdown (RNAi) of OsGRDP1 resulted in the lesion mimic phenotype. Moreover, the intact-protein level of OsGRDP1 was reduced in the spotted leaves from both overexpression and RNAi plants, suggesting that the disruption of intact OsGRDP1 is responsible for lesion formation. OsGRDP1 interacted with an aspartic proteinase, OsAP25. In the spl-D and overexpression plants, proteinase activity was elevated, and lesion formation was partially suppressed by an aspartic proteinase inhibitor. Taken together, our results reveal that OsGRDP1 is a critical feedback regulator, thus contributing to the elucidation of the mechanism underlying cell death and disease resistance.

Ectopic Expression of the Transcriptional Regulator silky3 Causes Pleiotropic Meristem and Sex Determination Defects in Maize Inflorescences.

Maize (Zea mays) is a monoecious plant, in which inflorescence morphogenesis involves complicated molecular regulatory mechanisms. Although many related genes have been cloned, our understanding of the molecular mechanism underlying maize inflorescence development remains limited. Here, we identified a maize semi-dominant mutant Silky3 (Si3), which displays pleiotropic defects during inflorescence development, including loss of determinacy and identity in meristems and floral organs, as well as the sexual transformation of tassel florets. We cloned the si3 gene using a map-based approach. Functional analysis reveals that SI3 is a nuclear protein and may act as a transcriptional regulator. Transcriptome analysis reveals that the ectopic expression of si3 strongly represses multiple biological processes, especially the flower development pathways. RNA in situ hybridization similarly shows that the expression patterns of genes responsible for flower development are changed in the Si3 mutant. In addition, the homeostasis of jasmonic acid and gibberellic acid are altered in the Si3 young tassels, and application of exogenous jasmonic acid can rescue the sex reversal phenotype of Si3 The defects we characterized in various regulatory pathways can explain the complex phenotypes of Si3 mutant, and this study deepens our knowledge of maize inflorescence development.

Analysis of Pyricularia oryzae and P. grisea from Different Hosts Based on Multilocus Phylogeny and Pathogenicity Associated with Host Preference in China.

Pyricularia oryzae and P. grisea are important agents of major diseases on a wide range of gramineous hosts. Whereas P. oryzae is the most important pathogen causing outbreaks of rice blast, P. grisea is mainly a pathogen of crabgrass. In this study, 103 P. oryzae and 20 P. grisea isolates were collected from seven species of plants, and we analyzed their phylogeny, pathogenicity, and relationship with host preferences to investigate the differences among them from different hosts. Based on phylogenetic analysis of multilocus sequences, 16 isolates from crabgrass and four isolates from green bristlegrass were identified as P. grisea and another 103 isolates from crabgrass, green bristlegrass, goose grass, foxtail millet, wild millet, rice, and sedge belonged to P. oryzae. Results of pathogenicity tests by artificial inoculation demonstrated that six of 10 P. oryzae isolates from rice and three of 44 P. oryzae isolates from green bristlegrass showed cross-infectivity on green bristlegrass and rice, respectively. Taken together, our results demonstrated that isolates from green bristlegrass and crabgrass consist of both P. oryzae and P. grisea and that P. oryzae isolates showed cross-infectivity between rice and green bristlegrass, suggesting that host shifts may occur for P. oryzae and P. grisea.

Glutamate synthase MoGlt1-mediated glutamate homeostasis is important for autophagy, virulence and conidiation in the rice blast fungus.

Glutamate homeostasis plays a vital role in central nitrogen metabolism and coordinates several key metabolic functions. However, its function in fungal pathogenesis and development has not been investigated in detail. In this study, we identified and characterized a glutamate synthase gene MoGLT1 in the rice blast fungus Magnaporthe oryzae that was important to glutamate homeostasis. MoGLT1 was constitutively expressed, but showed the highest expression level in appressoria. Deletion of MoGLT1 resulted in a significant reduction in conidiation and virulence. The ΔMoglt1 mutants were defective in appressorial penetration and the differentiation and spread of invasive hyphae in penetrated plant cells. The addition of exogenous glutamic acid partially rescued the defects of the ΔMoglt1 mutants in conidiation and plant infection. Assays for MoAtg8 expression and localization showed that the ΔMoglt1 mutants were defective in autophagy. The ΔMoglt1 mutants were delayed in the mobilization of glycogens and lipid bodies from conidia to developing appressoria. Taken together, our results show that glutamate synthase MoGlt1-mediated glutamate homeostasis is important for pathogenesis and development in the rice blast fungus, possibly via the regulation of autophagy.

Systematic analysis of the lysine acetylome in Fusarium graminearum.

BACKGROUND: Lysine acetylation in proteins is a ubiquitous and conserved post-translational modification, playing a critical regulatory role in almost every aspect of living cells. Although known for many years, its function remains elusive in Fusarium graminearum, one of the most important necrotrophic plant pathogens with huge economic impact. RESULTS: By the combination of affinity enrichment and high-resolution LC-MS/MS analysis, large-scale lysine acetylome analysis was performed. In total, 577 lysine acetylation sites matched to 364 different proteins were identified. Bioinformatics analysis of the acetylome showed that the acetylated proteins are involved in a wide range of cellular functions and exhibit diverse subcellular localizations. Remarkably, 10 proteins involved in the virulence or DON (deoxynivalenol) biosynthesis were found to be acetylated, including 4 transcription factors, 4 protein kinases and 2 phosphatases. Protein-protein interaction network analysis revealed that acetylated protein complexes are involved in diversified interactions. CONCLUSIONS: This work provides the first comprehensive survey of a possible lysine acetylome in F. graminearum and reveals previously unappreciated roles of lysine acetylation in the regulation of diverse biological processes. This work provides a resource for functional analysis of acetylated proteins in filamentous fungi.