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.

The Molecular Genetics and Genomics of Plant-Pathogen Interactions.

Plants have evolved an intricate immune system to protect themselves from potential pathogens [...].

The synthetic NLR RGA5HMA5 requires multiple interfaces within and outside the integrated domain for effector recognition.

Some plant sensor nucleotide-binding leucine-rich repeat (NLR) receptors detect pathogen effectors through their integrated domains (IDs). Rice RGA5 sensor NLR recognizes its corresponding effectors AVR-Pia and AVR1-CO39 from the blast fungus Magnaporthe oryzae through direct binding to its heavy metal-associated (HMA) ID to trigger the RGA4 helper NLR-dependent resistance in rice. Here, we report a mutant of RGA5 named RGA5HMA5 that confers complete resistance in transgenic rice plants to the M. oryzae strains expressing the noncorresponding effector AVR-PikD. RGA5HMA5 carries three engineered interfaces, two of which lie in the HMA ID and the other in the C-terminal Lys-rich stretch tailing the ID. However, RGA5 variants having one or two of the three interfaces, including replacing all the Lys residues with Glu residues in the Lys-rich stretch, failed to activate RGA4-dependent cell death of rice protoplasts. Altogether, this work demonstrates that sensor NLRs require a concerted action of multiple surfaces within and outside the IDs to both recognize effectors and activate helper NLR-mediated resistance, and has implications in structure-guided designing of sensor NLRs.

A gln-tRNA-based CRISPR/Cas9 knockout system enables the functional characterization of genes in the genetically recalcitrant brassica anthracnose fungus Colletotrichum higginsianum.

Colletotrichum higginsianum causes anthracnose disease in brassicas. The availability of the C. higginsianum genome has paved the way for the genome-wide exploration of genes associated with virulence/pathogenicity. However, delimiting the biological functions of these genes remains an arduous task due to the recalcitrance of C. higginsianum to genetic manipulations. Here, we report a CRISPR/Cas9-based system that can knock out the genes in C. higginsianum with a staggering 100% homologous recombination frequency (HRF). The system comprises two vectors: pCas9-Ch_tRp-sgRNA, in which a C. higginsianum glutaminyl-tRNA drives the expression of sgRNA, and pCE-Zero-HPT carrying a donor DNA cassette containing the marker gene HPT flanked by homology arms. Upon co-transformation of the C. higginsianum protoplasts, pCas9-Ch_tRp-sgRNA causes a DNA double-strand break in the targeted gene, followed by homology-directed replacement of the gene with HPT by pCE-Zero-HPT, thereby generating loss-of-function mutants. Using the system, we generated the knockout mutants of two effector candidates (ChBas3 and OBR06881) with a 100% HRF. Interestingly, the ΔChBas3 and ΔOBR06881 mutants did not seem to affect the C. higginsianum infection of Arabidopsis thaliana. Altogether, the CRISPR/Cas9 system developed in the study enables the targeted deletion of genes, including effectors, in C. higginsianum, thus determining their biological functions.

The GRAS protein OsDLA involves in brassinosteroid signalling and positively regulates blast resistance by forming a module with GSK2 and OsWRKY53 in rice.

Brassinosteroids (BRs) play a crucial role in shaping the architecture of rice (Oryza sativa) plants. However, the regulatory mechanism of BR signalling in rice immunity remains largely unexplored. Here we identify a rice mutant dla, which exhibits decreased leaf angles and is insensitive to 24-epiBL (a highly active synthetic BR), resembling the BR-deficient phenotype. The dla mutation caused by a T-DNA insertion in the OsDLA gene leads to downregulation of the causative gene. The OsDLA knockout plants display reduced leaf angles and less sensitivity to 24-epiBL. In addition, both dla mutant and OsDLA knockout plants are more susceptible to rice blast compared to the wild type. OsDLA is a GRAS transcription factor and interacts with the BR signalling core negative regulator, GSK2. GSK2 phosphorylates OsDLA for degradation via the 26S proteasome. The GSK2 RNAi line exhibits enhanced rice blast resistance, while the overexpression lines thereof show susceptibility to rice blast. Furthermore, we show that OsDLA interacts with and stabilizes the WRKY transcription factor OsWRKY53, which has been demonstrated to positively regulate BR signalling and blast resistance. OsWRKY53 directly binds the promoter of PBZ1 and activates its expression, and this activation can be enhanced by OsDLA. Together, our findings unravel a novel mechanism whereby the GSK2-OsDLA-OsWRKY53 module coordinates blast resistance and plant architecture via BR signalling in rice.

A dual-subcellular localized ß-glucosidase confers pathogen and insect resistance without a yield penalty in maize.

Maize is one of the most important crops for food, cattle feed and energy production. However, maize is frequently attacked by various pathogens and pests, which pose a significant threat to maize yield and quality. Identification of quantitative trait loci and genes for resistance to pests will provide the basis for resistance breeding in maize. Here, a ß-glucosidase ZmBGLU17 was identified as a resistance gene against Pythium aphanidermatum, one of the causal agents of corn stalk rot, by genome-wide association analysis. Genetic analysis showed that both structural variations at the promoter and a single nucleotide polymorphism at the fifth intron distinguish the two ZmBGLU17 alleles. The causative polymorphism near the GT-AG splice site activates cryptic alternative splicing and intron retention of ZmBGLU17 mRNA, leading to the downregulation of functional ZmBGLU17 transcripts. ZmBGLU17 localizes in both the extracellular matrix and vacuole and contribute to the accumulation of two defence metabolites lignin and DIMBOA. Silencing of ZmBGLU17 reduces maize resistance against P. aphanidermatum, while overexpression significantly enhances resistance of maize against both the oomycete pathogen P. aphanidermatum and the Asian corn borer Ostrinia furnacalis. Notably, ZmBGLU17 overexpression lines exhibited normal growth and yield phenotype in the field. Taken together, our findings reveal that the apoplastic and vacuolar localized ZmBGLU17 confers resistance to both pathogens and insect pests in maize without a yield penalty, by fine-tuning the accumulation of lignin and DIMBOA.

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.

An ERAD-related ubiquitin-conjugating enzyme boosts broad-spectrum disease resistance and yield in rice.

Rice is a staple crop for over half of the global population. However, blast disease caused by Magnaporthe orzae can result in more than a 30% loss in rice yield in epidemic years. Although some major resistance genes bolstering blast resistance have been identified in rice, their stacking in elite cultivars usually leads to yield penalties. Here we report that OsUBC45, a ubiquitin-conjugating enzyme functioning in the endoplasmic reticulum-associated protein degradation system, promotes broad-spectrum disease resistance and yield in rice. OsUBC45 is induced upon infection by M. oryzae, and its overexpression enhances resistance to blast disease and bacterial leaf blight by elevating pathogen-associated molecular pattern-triggered immunity (PTI) while nullifying the gene-attenuated PTI. The OsUBC45 overexpression also increases grain yield by over 10%. Further, OsUBC45 enhances the degradation of glycogen synthase kinase 3 OsGSK3 and aquaporin OsPIP2;1, which negatively regulate the grain size and PTI, respectively. The OsUBC45 reported in our study has the potential for improving yield and disease resistance for sustainable rice production.

CRISPR/Cas9-mediated deletion of large chromosomal segments identifies a minichromosome modulating the Colletotrichum graminicola virulence on maize.

Colletotrichum graminicola causes anthracnose on maize, an economically significant disease worldwide. To decipher how the pathogen controls its virulence/pathogenicity on maize at the minichromosomal level, we sequenced the genome and transcriptome of the C. graminicola strain T1-3-3. The 61.91 Mb genome contains three transcriptionally repressed, full-length strain-specific minichromosomes (<1 Mb; Chr11 through Chr13). A CRISPR/Cas9-based system was developed to knock out large chromosomal segments; it involved the generation of multiple simultaneous DNA double-strand breaks across a targeted genomic region, followed by homology-directed replacement thereof with a donor DNA template carrying the selectable marker hygromycin phosphotransferase gene flanked by homologous sequence arms of the targeted region. Using this system, we obtained distinct mutants functionally nullisomic for individual minichromosomes. Only the ΔChr12 mutant lacking the 498.44 Kb genomic region carrying all of the 31 genes of Chr12 exhibited attenuated virulence on maize and was indistinguishable from T1-3-3 in fungal growth and conidiation, indicating that Chr12 is a conditionally dispensable minichromosome and imparts full virulence to C. graminicola on maize. The CRISPR/Cas9-mediated genome editing system developed in this study will enable the determination of the biological functions of minichromosomes or large chromosomal segments in fungal plant pathogens.

Magnaporthe oryzae endoplasmic reticulum membrane complex regulates the biogenesis of membrane proteins for pathogenicity.

In eukaryotes, the majority of newly synthesized integral membrane proteins are inserted into the endoplasmic reticulum (ER) membrane before transferred to their functional sites. The conserved ER membrane complex (EMC) takes part in the insertion process for tail-anchored membrane proteins. However, the function of EMC in phytopathogenic fungi has not been characterized. Here, we report the identification and functional characterization of two EMC subunits MoEmc5 and MoEmc2 in Magnaporthe oryzae. The knockout mutants ΔMoemc5 and ΔMoemc2 exhibit substantial defect in autophagy, pathogenicity, cell wall integrity, and magnesium ion sensitivity. We demonstrate that the autophagy process was severely impaired in the ΔMoemc5 and ΔMoemc2 mutants because of the low-protein steady-state level of Atg9, the sole membrane-associated autophagy protein. Furthermore, the protein level of membrane proteins Chs4, Fks1, and MoMnr2 is also significantly reduced in the ΔMoemc5 and ΔMoemc2 strains, leading to their supersensitivity to Calcofluor white, Congo red, and magnesium. In addition, MoEmc5, but not MoEmc2, acts as a magnesium transporter independent of its EMC function. Magnaporthe oryzae EMC regulates the biogenesis of membrane proteins for autophagy and virulence; therefore, EMC subunits could be potential targets for fungicide design in the future.

Two Species of Stemphylium, S. astragali and S. henanense sp. nov., Causing Leaf and Stem Spot Disease on Astragalus sinicus in China.

Astragalus sinicus is a versatile legume crop, primarily utilized as a green manure in China. During 2020 and 2021, A. sinicus plants exhibiting dark brown or reddish-brown lesions or spots on leaves and stems were collected from fields in the Henan, Sichuan, and Guangxi provinces of China. Sixteen single-spore isolates were isolated from the infected leaf and stem tissue samples. Phylogenetic analyses based on the concatenated internal transcribed spacer, gapdh, and cmdA sequences indicated that 14 of them belong to Stemphylium astragali, whereas two isolates can be well separated from other known species in this genus. Based on the morphological characteristics and nucleotide polymorphisms with sister taxa, the two isolates were identified as a new species named S. henanense. Furthermore, pathogenicity assays showed that the S. astragali and S. henanense isolates caused leaf and stem spot symptoms on A. sinicus. Altogether, we describe a new species of Stemphylium (i.e., S. henanense sp. nov.) causing leaf spot disease of A. sinicus. In addition, this is the first report of S. astragali causing stem spot disease of A. sinicus.

The Hidden Truths of Fungal Virulence and Adaptation on Hosts: Unraveling the Conditional Dispensability of Minichromosomes in the Hemibiotrophic Colletotrichum Pathogens.

Colletotrichum spp. are ascomycete fungi and cause anthracnose disease in numerous crops of economic significance. The genomes of these fungi are distributed among ten core chromosomes and two to three minichromosomes. While the core chromosomes regulate fungal growth, development and virulence, the extent to which the minichromosomes are involved in these processes is still uncertain. Here, we discuss the minichromosomes of three hemibiotrophic Colletotrichum pathogens, i.e., C. graminicola, C. higginsianum and C. lentis. These minichromosomes are typically less than one megabase in length, characterized by containing higher repetitive DNA elements, lower GC content, higher frequency of repeat-induced point mutations (RIPMs) and sparse gene distribution. Molecular genetics and functional analyses have revealed that these pathogens harbor one conditionally dispensable minichromosome, which is dispensable for fungal growth and development but indispensable for fungal virulence on hosts. They appear to be strain-specific innovations and are highly compartmentalized into AT-rich and GC-rich blocks, resulting from RIPMs, which may help protect the conditionally dispensable minichromosomes from erosion of already scarce genes, thereby helping the Colletotrichum pathogens maintain adaptability on hosts. Overall, understanding the mechanisms underlying the conditional dispensability of these minichromosomes could lead to new strategies for controlling anthracnose disease in crops.

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.

Gapless reference genome assembly of Didymella glomerata, a new fungal pathogen of maize causing Didymella leaf blight.

Didymella leaf blight (DLB) caused by Didymella glomerata is a new fungal disease of maize (Zea mays), first detected in 2021 in Panjin, Liaoning province of China. Here we report the reference genome assembly of D. glomerata to unravel how the fungal pathogen controls its virulence on maize at the molecular level. A maize-infecting strain Pj-2 of the pathogen was sequenced on the Illumina NovaSeq 6000 and PacBio Sequel II platforms at a 575-fold genomic coverage. The 33.17 Mb gapless genome assembly comprises 32 scaffolds with L/N50 of 11/1.36 Mb, four of which represent full-length chromosomes. The Pj-2 genome is predicted to contain 10,334 protein-coding genes, of which 211, 12 and 134 encode effector candidates, secondary metabolite backbone-forming enzymes and CAZymes, respectively. Some of these genes are potentially implicated in niche adaptation and expansion, such as colonizing new hosts like maize. Phylogenomic analysis of eight strains of six Didymella spp., including three sequenced strains of D. glomerata, reveals that the maize (Pj-2)- and Chrysanthemum (CBS 528.66)-infecting strains of D. glomerata are genetically similar (sharing 92.37% genome with 98.89% identity), whereas Pj-2 shows truncated collinearity with extensive chromosomal rearrangements with the Malus-infecting strain M27-16 of D. glomerata (sharing only 55.01% genome with 88.20% identity). Pj-2 and CBS 528.66 carry four major reciprocal translocations in their genomes, which may enable them to colonize the different hosts. Furthermore, germplasm screening against Pj-2 led to the identification of three sources of DLB resistance in maize, including a tropical inbred line CML496. DLB resistance in the line is attributed to the accumulation of ROS H2O2 in the apoplastic space of the infected cells, which likely restricts the fungal growth and proliferation.

Molecular Genetics of Anthracnose Resistance in Maize.

Maize (Zea mays), also called corn, is one of the top three staple food crops worldwide and is also utilized as feed (e.g., feed grain and silage) and a source of biofuel (e.g., bioethanol). Maize production is hampered by a myriad of factors, including although not limited to fungal diseases, which reduce grain yield and downgrade kernel quality. One such disease is anthracnose leaf blight and stalk rot (ALB and ASR) caused by the hemibiotrophic fungal pathogen Colletotrichum graminicola. The pathogen deploys a biphasic infection strategy to colonize susceptible maize genotypes, comprising latent (symptomless) biotrophic and destructive (symptomatic) necrotrophic phases. However, the resistant maize genotypes restrict the C. graminicola infection and in planta fungal proliferation during the biotrophic phase of the infection. Some studies on the inheritance of ASR resistance in the populations derived from biparental resistant and susceptible genotypes reveal that anthracnose is likely a gene-for-gene disease in which the resistant maize genotypes and C. graminicola recognize each other by their matching pairs of nucleotide-binding leucine-rich repeat resistance (NLR) proteins (whose coding genes are localized in disease QTL) and effectors (1-2 effectors/NLR) during the biotrophic phase of infection. The Z. mays genome encodes approximately 144 NLRs, two of which, RCg1 and RCg1b, located on chromosome 4, were cloned and functionally validated for their role in ASR resistance. Here, we discuss the genetic architecture of anthracnose resistance in the resistant maize genotypes, i.e., disease QTL and underlying resistance genes. In addition, this review also highlights the disease cycle of C. graminicola and molecular factors (e.g., virulence/pathogenicity factors such as effectors and secondary metabolites) that contribute to the pathogen's virulence on maize. A detailed understanding of molecular genetics underlying the maize-C. graminicola interaction will help devise effective management strategies against ALB and ASR.

First Report of Didymella glomerata Causing Didymella Leaf Blight on Maize in China.

Maize (Zea mays L.) is a staple food crop worldwide. In July 2021, gray leaf blight was observed on maize leaves in a field located in Panjin (41°7'11.98" N, 122°4'14.57" E), Liaoning Province, China. Nearly 5% of the maize plants were affected in the field. The leaves of the affected plants showed oval to oblong, gray, sunken lesions with yellow or tan margins. The lesions were scattered all over the leaf surface; however, they were absent on the stalks and other parts of the affected plants. To isolate the pathogen, leaf discs (1.25 mm2) excised from the blight lesions were surface-sterilized with 70% ethanol for 30 seconds, followed by 20% NaOCl for 2 minutes and finally rinsed three times with sterilized water. The discs were cultured on potato dextrose agar (PDA) plates supplemented with streptomycin (100 mg/L) and incubated at 25oC under a 12-h photoperiod for 7 days. Six single spore isolates (two per sampled infected leaf) were purified from the PDA culture plates. The fungal colonies of three selected isolates (one per sampled infected leaf; Pj-1, Pj-2, and Pj-3) were dark brown on the PDA plates and devoid of aerial hyphae; all three isolates grew 11 mm/day on the PDA plates. The number of conidia produced by the isolates on the 6-cm PDA plates 7 days after incubation was ranged from 160 x 108 to 208 x 108 (n = 36). Conidia were hyaline, single-celled and ellipsoidal (3.35-3.56 µm [width] x 6.47-6.70 [length] µm; n = 36). To identify the pathogen, four loci, i.e., 28S subunit (large subunit [LSU]) of the nuclear ribosomal (nr) DNA, internal transcribed spacer (ITS) region (ITS1, 5.8S subunit of nrDNA, and ITS2), the second-largest subunit of RNA polymerase II (rpb2) and ß-tubulin (tub2) were amplified using the primer sets described in the study by Chen el al. 2015. BLASTn search against GenBank revealed that the four amplicon sequences originating from Pj-1, Pj-2, and Pj-3 showed 99-100% homology to the type strain CBS 528.66 of D. glomerata. A phylogenetic tree deduced from a maximum likelihood analysis of a concatenated MUSCLE-based alignment of LSU, ITS region, rpb2, and tub2 sequences of 12 isolates/strains showed that the Pj isolates clustered together with CBS 528.66, along with other D. glomerata isolates/strains, with a high bootstrap support value (i.e., 99). Based on both morphological characteristics and molecular phylogeny, Pj-1, Pj-2, and Pj-3 were identified as the D. glomerata isolates. Since the amplicon sequences of the three isolates were identical, only Pj-2 sequences were deposited in GenBank with accession numbers OM372474 (LSU), OK485138 (ITS), OM406188 (rpb2), and OK485135 (tub2). To confirm pathogenicity, 14-day-old plants (V3 growth stage) of a maize cultivar P178 were spray-inoculated with the Pj-2 conidia (1 x 107 conidia/mL) in a growth chamber. The inoculated leaves exhibited typical gray leaf blight lesions (similar to those detected in the maize field) 7 days post-inoculation at 25oC and 95-100% humidity under a 12-h photoperiod, whereas the leaves spray-inoculated with sterilized water remained healthy. The pathogenicity assay was repeated three times; the pathogen was re-isolated from the inoculated leaves each time and confirmed by the morphological characteristics and the molecular phylogeny based on the four loci to be D. glomerata, fulfilling Koch's postulates. This first report of D. glomerata causing Didymella leaf blight on maize will help develop robust disease management strategies against this emerging fungal pathogen.

MoSnf5 Regulates Fungal Virulence, Growth, and Conidiation in Magnaporthe oryzae.

Snf5 (sucrose nonfermenting) is a core component of the SWI/SNF complexes and regulates diverse cellular processes in model eukaryotes. In plant pathogenic fungi, its biological function and underlying mechanisms remain unexplored. In this study, we investigated the biological roles of MoSnf5 in plant infection and fungal development in the rice blast pathogen Magnaporthe oryzae. The gene deletion mutants of MoSNF5 exhibited slower vegetative hyphal growth, severe defects in conidiogenesis, and impaired virulence and galactose utilization capacities. Domain dissection assays showed that the Snf5 domain and the N- and C-termini of MoSnf5 were all required for its full functions. Co-immunoprecipitation and yeast two-hybrid assays showed that MoSnf5 physically interacts with four proteins, including a transcription initiation factor MoTaf14. Interestingly, the ∆MoTaf14 mutants showed similar phenotypes as the ∆Mosnf5 mutants on fungal virulence and development. Moreover, assays on GFP-MoAtg8 expression and localization showed that both the ∆Mosnf5 and ∆MoTaf14 mutants were defective in autophagy. Taken together, MoSnf5 regulates fungal virulence, growth, and conidiation, possibly through regulating galactose utilization and autophagy in M. oryzae.

A designer rice NLR immune receptor confers resistance to the rice blast fungus carrying noncorresponding avirulence effectors.

Plant nucleotide-binding and leucine-rich repeat (NLR) receptors recognize avirulence effectors directly through their integrated domains (IDs) or indirectly via the effector-targeted proteins. Previous studies have succeeded in generating designer NLR receptors with new recognition profiles by engineering IDs or targeted proteins based on prior knowledge of their interactions with the effectors. However, it is yet a challenge to design a new plant receptor capable of recognizing effectors that function by unknown mechanisms. Several rice NLR immune receptors, including RGA5, possess an integrated heavy metal-associated (HMA) domain that recognizes corresponding Magnaporthe oryzae Avrs and ToxB-like (MAX) effectors in the rice blast fungus. Here, we report a designer rice NLR receptor RGA5HMA2 carrying an engineered, integrated HMA domain (RGA5-HMA2) that can recognize the noncorresponding MAX effector AvrPib and confers the RGA4-dependent resistance to the M. oryzae isolates expressing AvrPib, which originally triggers the Pib-mediated blast resistance via unknown mechanisms. The RGA5-HMA2 domain is contrived based on the high structural similarity of AvrPib with two MAX effectors, AVR-Pia and AVR1-CO39, recognized by cognate RGA5-HMA, the binding interface between AVR1-CO39 and RGA5-HMA, and the distinct surface charge of AvrPib and RAG5-HMA. This work demonstrates that rice NLR receptors with the HMA domain can be engineered to confer resistance to the M. oryzae isolates noncorresponding but structurally similar MAX effectors, which manifest cognate NLR receptor-mediated resistance with unknown mechanisms. Our study also provides a practical approach for developing rice multilines and broad race spectrum-resistant cultivars by introducing a series of engineered NLR receptors.

Transcriptional Landscapes of Long Non-coding RNAs and Alternative Splicing in Pyricularia oryzae Revealed by RNA-Seq.

Pyricularia oryzae causes the rice blast, which is one of the most devastating crop diseases worldwide, and is a model fungal pathogen widely used for dissecting the molecular mechanisms underlying fungal virulence/pathogenicity. Although the whole genome sequence of P. oryzae is publicly available, its current transcriptomes remain incomplete, lacking the information on non-protein coding genes and alternative splicing. Here, we performed and analyzed RNA-Seq of conidia and hyphae, resulting in the identification of 3,374 novel genes. Interestingly, the vast majority of these novel genes likely transcribed long non-coding RNAs (lncRNAs), and most of them were localized in the intergenic regions. Notably, their expressions were concomitant with the transcription of neighboring genes thereof in conidia and hyphae. In addition, 2,358 genes were found to undergo alternative splicing events. Furthermore, we exemplified that a lncRNA was important for hyphal growth likely by regulating the neighboring protein-coding gene and that alternative splicing of the transcription factor gene CON7 was required for appressorium formation. In summary, results from this study indicate that lncRNA transcripts and alternative splicing events are two important mechanisms for regulating the expression of genes important for conidiation, hyphal growth, and pathogenesis, and provide new insights into transcriptomes and gene regulation in the rice blast fungus.