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The ubiquitin-proteasome system (UPS) plays crucial roles in cellular processes including plant growth, development, and stress responses. In this study, we report that a pair of E3 ubiquitin ligases, AvrPiz-t-interaction protein 6 (APIP6) and IPA1-interaction protein 1 (IPI1), intricately target early flowering3 (ELF3) paralogous proteins to control rice immunity and flowering. APIP6 forms homo-oligomers or hetero-oligomers with IPI1. Both proteins interact with OsELF3-2, promoting its degradation to positively control resistance against the rice blast fungus (Magnaporthe oryzae). Intriguingly, overexpression of IPI1 in Nipponbare caused significantly late-flowering phenotypes similar to the oself3-1 mutant. Except for late flowering, oself3-1 enhances resistance against M. oryzae. IPI1 also interacts with and promotes the degradation of OsELF3-1, a paralog of OsELF3-2. Notably, IPI1 and APIP6 synergistically modulate OsELF3s degradation, finely tuning blast disease resistance by targeting OsELF3-2, while IPI1 controls both disease resistance and flowering by targeting OsELF3-1. This study unravels multiple functions for a pair of E3 ligases in rice., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Soil salinization leads to a reduction in arable land area, which seriously endangers food security. Developing saline-alkali land has become a key measure to address the contradiction between population growth and limited arable land. Rice is the most important global food crop, feeding half of the world's population and making it a suitable choice for planting on saline-alkali lands. The traditional salt-alkali improvement method has several drawbacks. Currently, non-thermal plasma (NTP) technology is being increasingly applied in agriculture. However, there are few reports on the cultivation of salt/alkali-tolerant rice. Under alkaline stress, argon NTP treatment significantly increased the germination rate of Longdao 5 (LD5) rice seeds. In addition, at 15 kV and 120 s, NTP treatment significantly increased the activity of antioxidant enzymes such as catalase and SOD. NTP treatment induced changes in genes related to salt-alkali stress in rice seedlings, such as chitinase and xylanase inhibitor proteins, which increased the tolerance of the seeds to salt-alkali stress. This experiment has expanded the application scope of NTP in agriculture, providing a more cost-effective, less harmful, and faster method for developing salt-alkali-tolerant rice and laying a theoretical foundation for cultivating NTP-enhanced salt-alkali-tolerant rice., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Broad-spectrum disease resistance (BSR) is crucial for controlling plant diseases and relies on immune signals that are subject to transcriptional and post-translational regulation. How plants integrate and coordinate these signals remains unclear. We show here that the rice really interesting new gene (RING)-type E3 ubiquitin ligase OsRING113 targets APIP5, a negative regulator of plant immunity and programmed cell death (PCD), for 26S proteasomal degradation. The osring113 mutants in Nipponbare exhibited decreased BSR, while the overexpressing OsRING113 plants showed enhanced BSR against Magnaporthe oryzae (M. oryzae) and Xanthomonas oryzae pv. oryzae (Xoo). Furthermore, APIP5 directly suppressed the transcription of the Bowman-Birk trypsin inhibitor genes OsBBTI5 and AvrPiz-t-interacting protein 4 (APIP4). Overexpression of these two genes, which are partially required for APIP5-mediated PCD and disease resistance, conferred BSR. OsBBTI5 and APIP4 associated with and stabilized the pathogenesis-related protein OsPR1aL, which promotes M. oryzae resistance. Our results identify an immune module with integrated and coordinated hierarchical regulations that confer BSR in plants., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Plants typically activate distinct defense pathways against various pathogens. Heightened resistance to one pathogen often coincides with increased susceptibility to another pathogen. However, the underlying molecular basis of this antagonistic response remains unclear. Here, we demonstrate that mutants defective in the transcription factor ETHYLENE-INSENSITIVE 3-LIKE 2 (OsEIL2) exhibited enhanced resistance to the biotrophic bacterial pathogen Xanthomonas oryzae pv oryzae and to the hemibiotrophic fungal pathogen Magnaporthe oryzae, but enhanced susceptibility to the necrotrophic fungal pathogen Rhizoctonia solani. Furthermore, necrotroph-induced OsEIL2 binds to the promoter of OsWRKY67 with high affinity, leading to the upregulation of salicylic acid (SA)/jasmonic acid (JA) pathway genes and increased SA/JA levels, ultimately resulting in enhanced resistance. However, biotroph- and hemibiotroph-induced OsEIL2 targets OsERF083, resulting in the inhibition of SA/JA pathway genes and decreased SA/JA levels, ultimately leading to reduced resistance. Our findings unveil a previously uncharacterized defense mechanism wherein two distinct transcriptional regulatory modules differentially mediate immunity against pathogens with different lifestyles through the transcriptional reprogramming of phytohormone pathway genes., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Although the antagonistic effects of host resistance against biotrophic and necrotrophic pathogens have been documented in various plants, the underlying mechanisms are unknown. Here, we investigated the antagonistic resistance mediated by the transcription factor ETHYLENE-INSENSITIVE3-LIKE 3 (OsEIL3) in rice. The Oseil3 mutant confers enhanced resistance to the necrotroph Rhizoctonia solani but greater susceptibility to the hemibiotroph Magnaporthe oryzae and biotroph Xanthomonas oryzae pv. oryzae. OsEIL3 directly activates OsERF040 transcription while repressing OsWRKY28 transcription. The infection of R. solani and M. oryzae or Xoo influences the extent of binding of OsEIL3 to OsWRKY28 and OsERF040 promoters, resulting in the repression or activation of both salicylic acid (SA)- and jasmonic acid (JA)-dependent pathways and enhanced susceptibility or resistance, respectively. These results demonstrate that the distinct effects of plant immunity to different pathogen types are determined by two transcription factor modules that control transcriptional reprogramming and the SA and JA pathways., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

F-box protein is a subunit of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which plays a critical role in regulating different pathways in plant immunity. In this study, we identified the rice (Oryza sativa) F-box protein OsFBX156, which targets the heat shock protein 70 (OsHSP71.1) to regulate resistance to the rice blast fungus Magnaporthe oryzae. Overexpression of OsFBX156 or knockout of OsHSP71.1 in rice resulted in the elevation of pathogenesis-related (PR) genes and an induction burst of reactive oxygen species (ROS) after flg22 and chitin treatments, thereby enhancing resistance to M. oryzae. Furthermore, OsFBX156 can promote the degradation of OsHSP71.1 through the 26S proteasome pathway. This study sheds lights on a novel mechanism wherein the F-box protein OsFBX156 targets OsHSP71.1 for degradation to promote ROS production and PR gene expression, thereby positively regulating rice innate immunity., (© 2024 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Proteins containing a ubiquitin regulatory X (UBX) domain are cofactors of Cell Division Cycle 48 (CDC48) and function in protein quality control. However, whether and how UBX-containing proteins participate in host-microbe interactions remain unclear. Here we show that MoNLE1, an effector from the fungal pathogen Magnaporthe oryzae, is a core virulence factor that suppresses rice immunity by specifically interfering with OsPUX8B.2. The UBX domain of OsPUX8B.2 is required for its binding to OsATG8 and OsCDC48-6 and controls its 26 S proteasome-dependent stability. OsPUX8B.2 and OsCDC48-6 positively regulate plant immunity against blast fungus, while the high-temperature tolerance heat-shock protein OsBHT, a putative cytoplasmic substrate of OsPUX8B.2-OsCDC48-6, negatively regulates defense against blast infection. MoNLE1 promotes the nuclear migration and degradation of OsPUX8B.2 and disturbs its association with OsBHT. Given the high conservation of MoNLE1 among fungal isolates, plants with broad and durable blast resistance might be generated by engineering intracellular proteins resistant to MoNLE1., (© 2024. The Author(s).)

Lesion mimic mutations (LMMs) often confer broad-spectrum resistance (BSR) in plants, but with significant yield penalties. Sha et al. recently demonstrated that genome editing of the rice BSR gene RESISTANCE TO BLAST1 (RBL1), encoding a cytidine diphosphate diacylglycerol (CDP-DAG) synthase involved in phospholipid biosynthesis, confers multipathogen resistance without an obvious trade-off in yield., Competing Interests: Declaration of interests The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Long non-coding RNAs (lncRNAs) play critical roles in various biological processes in plants. Extensive studies utilizing high-throughput RNA sequencing have revealed that many lncRNAs are involved in plant disease resistance. Oryza sativa RNase P protein 30 (OsRpp30) has been identified as a positive regulator of rice immunity against fungal and bacterial pathogens. Nevertheless, the specific functions of lncRNAs in relation to OsRpp30-mediated disease resistance in rice remain elusive., Results: We conducted a comprehensive analysis of lncRNAs, miRNAs, and mRNAs expression patterns in wild type (WT), OsRpp30 overexpression (OsRpp30-OE), and OsRpp30 knockout (OsRpp30-KO) rice plants. In total, we identified 91 differentially expressed lncRNAs (DElncRNAs), 1671 differentially expressed mRNAs (DEmRNAs), and 41 differentially expressed miRNAs (DEmiRNAs) across the different rice lines. To gain further insights, we investigated the interaction between DElncRNAs and DEmRNAs, leading to the discovery of 10 trans- and 27 cis-targeting pairs specific to the OsRpp30-OE and OsRpp30-KO samples. In addition, we constructed a competing endogenous RNA (ceRNA) network comprising differentially expressed lncRNAs, miRNAs, and mRNAs to elucidate their intricate interplay in rice disease resistance. The ceRNA network analysis uncovered a set of gene targets regulated by lncRNAs and miRNAs, which were found to be involved in pathogen recognition, hormone pathways, transcription factor activation, and other biological processes related to plant immunity., Conclusions: Our study provides a comprehensive expression profiling of lncRNAs, miRNAs, and mRNAs in a collection of defense mutants in rice. To decipher the putative functional significance of lncRNAs, we constructed trans- and cis-targeting networks involving differentially expressed lncRNAs and mRNAs, as well as a ceRNA network incorporating differentially expressed lncRNAs, miRNAs, and mRNAs. Together, the findings from this study provide compelling evidence supporting the pivotal roles of lncRNAs in OsRpp30-mediated disease resistance in rice., (© 2023. The Author(s).)

Traditional rice blast resistance breeding largely depends on utilizing typical resistance (R) genes. However, the lack of durable R genes has prompted rice breeders to find new resistance resources. Susceptibility (S) genes are potential new targets for resistance genetic engineering using genome-editing technologies, but identifying them is still challenging. Here, through the integration of genome-wide association study (GWAS) and transcriptional analysis, we identified two genes, RNG1 and RNG3, whose polymorphisms in 3'-untranslated regions (3'-UTR) affected their expression variations. These polymorphisms could serve as molecular markers to identify rice blast-resistant accessions. Editing the 3'-UTRs using CRISPR/Cas9 technology affected the expression levels of two genes, which were positively associated with rice blast susceptibility. Knocking out either RNG1 or RNG3 in rice enhanced the rice blast and bacterial blight resistance, without impacting critical agronomic traits. RNG1 and RNG3 have two major genotypes in diverse rice germplasms. The frequency of the resistance genotype of these two genes significantly increased from landrace rice to modern cultivars. The obvious selective sweep flanking RNG3 suggested it has been artificially selected in modern rice breeding. These results provide new targets for S gene identification and open avenues for developing novel rice blast-resistant materials., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

During effector-triggered immunity (ETI) against the devastating rice blast pathogen Magnaporthe oryzae, Pi9 functions as an intracellular resistance protein sensing the pathogen-secreted effector AvrPi9 in rice. Importantly, the underlying recognition mechanism(s) between Pi9 and AvrPi9 remains elusive. In this study, we identified a rice ubiquitin-like domain-containing protein (UDP), AVRPI9-INTERACTING PROTEIN 1 (ANIP1), which is directly targeted by AvrPi9 and also binds to Pi9 in plants. Phenotypic analysis of anip1 mutants and plants overexpressing ANIP1 revealed that ANIP1 negatively modulates rice basal defense against M. oryzae. ANIP1 undergoes 26S proteasome-mediated degradation, which can be blocked by both AvrPi9 and Pi9. Moreover, ANIP1 physically associates with the rice WRKY transcription factor OsWRKY62, which also interacts with AvrPi9 and Pi9 in plants. In the absence of Pi9, ANIP1 negatively regulates OsWRKY62 abundance, which can be promoted by AvrPi9. Accordingly, knocking out of OsWRKY62 in a non-Pi9 background decreased immunity against M. oryzae. However, we also observed that OsWRKY62 plays negative roles in defense against a compatible M. oryzae strain in Pi9-harboring rice. Pi9 binds to ANIP1 and OsWRKY62 to form a complex, which may help to keep Pi9 in an inactive state and weaken rice immunity. Furthermore, using competitive binding assays, we showed that AvrPi9 promotes Pi9 dissociation from ANIP1, which could be an important step toward ETI activation. Taken together, our results reveal an immune strategy whereby a UDP-WRKY module, targeted by a fungal effector, modulates rice immunity in distinct ways in the presence or absence of the corresponding resistance protein., (Copyright © 2023 The Author. Published by Elsevier Inc. All rights reserved.)

Receptor-like kinases (RLKs) may initiate signaling pathways by perceiving and transmitting environmental signals to cellular machinery and play diverse roles in plant development and stress responses. The rice genome encodes more than one thousand RLKs, but only a small number have been characterized as receptors for phytohormones, polypeptides, elicitors, and effectors. Here, we screened the function of 11 RLKs in rice resistance to the blast fungus Magnaporthe oryzae ( M. oryzae ) and identified a negative regulator named BDR1 (Blast Disease Resistance 1). The expression of BDR1 was rapidly increased under M. oryzae infection, while silencing or knockout of BDR1 significantly enhanced M. oryzae resistance in two rice varieties. Protein interaction and kinase activity assays indicated that BDR1 directly interacted with and phosphorylated mitogen-activated kinase 3 (MPK3). Knockout of BDR1 compromised M. oryzae -induced MPK3 phosphorylation levels. Moreover, transcriptome analysis revealed that M. oryzae -elicited jasmonate (JA) signaling and terpenoid biosynthesis pathway were negatively regulated by BDR1 and MPK3. Mutation of JA biosynthetic ( allene oxide cyclase ( AOC )/signaling ( MYC2 ) genes decreased rice resistance to M. oryzae. Besides diterpenoid, the monoterpene linalool and the sesquiterpene caryophyllene were identified as unique defensive compounds against M. oryzae , and their biosynthesis genes ( TPS3 and TPS29 ) were transcriptionally regulated by JA signaling and suppressed by BDR1 and MPK3. These findings demonstrate the existence of a BDR1-MPK3 cascade that negatively mediates rice blast resistance by affecting JA-related defense responses.

Plant metabolites are critical components of immune signaling pathways; however, how these small molecules contribute to plant immunity remains largely elusive. Emerging evidence demonstrates that the rice nucleotide-binding leucine-rich repeat receptor (NLR)-interacting proteins regulate the biosynthesis of ethylene, hydroxycinnamoylputrescines and diterpenoid phytoalexins to modulate plant immunity., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Rice blast and bacterial blight, caused by the fungus Magnaporthe oryzae and the bacterium Xanthomonas oryzae pv. oryzae (Xoo), respectively, are devastating diseases affecting rice. Here, we report that a rice valine-glutamine (VQ) motif-containing protein, OsVQ25, balances broad-spectrum disease resistance and plant growth by interacting with a U-Box E3 ligase, OsPUB73, and a transcription factor, OsWRKY53. We show that OsPUB73 positively regulates rice resistance against M. oryzae and Xoo by interacting with and promoting OsVQ25 degradation via the 26S proteasome pathway. Knockout mutants of OsVQ25 exhibit enhanced resistance to both pathogens without a growth penalty. Furthermore, OsVQ25 interacts with and suppresses the transcriptional activity of OsWRKY53, a positive regulator of plant immunity. OsWRKY53 downstream defense-related genes and brassinosteroid signaling genes are upregulated in osvq25 mutants. Our findings reveal a ubiquitin E3 ligase-VQ protein-transcription factor module that fine-tunes plant immunity and growth at the transcriptional and posttranslational levels., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Phenolamide (PA) metabolites play important roles in the interaction between plants and pathogens. The putrescine hydroxycinnamoyl transferase genes OsPHT3 and OsPHT4 positively regulate rice cell death and resistance to Magnaporthe oryzae. The bZIP transcription factor APIP5, a negative regulator of cell death and rice immunity, directly binds to the OsPHT4 promoter to regulate putrescine-derived PAs. Whether other hydroxycinnamoyl transferase (HT) genes also participate in APIP5-mediated immunity remains unclear. Surprisingly, we find that genes encoding agmatine hydroxycinnamoyl transferases OsAHT1 and OsAHT2, tryptamine hydroxycinnamoyl transferases OsTBT1 and OsTBT2, and tyramine hydroxycinnamoyl transferases OsTHT1 and OsTHT2, responsible for the biosynthesis of polyamine-derived PAs are all up-regulated in APIP5-RNAi transgenic plants compared with segregated wild-type rice. Furthermore, both OsAHT1/2 and OsTBT1/2 are induced during M. oryzae infection, showing expression patterns similar to those previously reported for OsTHT1/2 and OsPHT3/4. Transgenic plants overexpressing either OsAHT2-GFP or OsTBT1-GFP show enhanced resistance against M. oryzae and accumulated more PA metabolites and lignin compared with wild-type plants. Interestingly, as demonstrated for OsPHT4, APIP5 directly binds to the promoters of OsAHT1/2, OsTBT1/2, and OsTHT1/2, repressing their transcription. Together, these results indicate that the HT genes are common targets of APIP5 and that PAs play critical roles in rice immunity., Competing Interests: Conflict of interest The authors have declared no competing interests., (Copyright © 2022 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. All rights reserved.)

Background: Ubiquitination is essential for many cellular processes in eukaryotes, including 26S proteasome-dependent protein degradation, cell cycle progression, transcriptional regulation, and signal transduction. Although numerous ubiquitinated proteins have been empirically identified, their cognate ubiquitin E3 ligases remain largely unknown., Results: Here, we generate a complete ubiquitin E3 ligase-encoding open reading frames (UbE3-ORFeome) library containing 98.94% of the 1515 E3 ligase genes in the rice (Oryza sativa L.) genome. In the test screens with four known ubiquitinated proteins, we identify both known and new E3s. The interaction and degradation between several E3s and their substrates are confirmed in vitro and in vivo. In addition, we identify the F-box E3 ligase OsFBK16 as a hub-interacting protein of the phenylalanine ammonia lyase family OsPAL1-OsPAL7. We demonstrate that OsFBK16 promotes the degradation of OsPAL1, OsPAL5, and OsPAL6. Remarkably, we find that overexpression of OsPAL1 or OsPAL6 as well as loss-of-function of OsFBK16 in rice displayed enhanced blast resistance, indicating that OsFBK16 degrades OsPALs to negatively regulate rice immunity., Conclusions: The rice UbE3-ORFeome is the first complete E3 ligase library in plants and represents a powerful proteomic resource for rapid identification of the cognate E3 ligases of ubiquitinated proteins and establishment of functional E3-substrate interactome in plants., (© 2022. The Author(s).)

WRKY transcription factors (TFs) play crucial roles in biotic and abiotic stress responses. However, their roles in thermal response are still largely elusive, especially in rice. In this study, we revealed the functions of WRKY10 TF and VQ8 protein containing VQ motif in rice thermotolerance. Overexpression of WRKY10 or loss of VQ8 function increases thermosensitivity, whereas conversely, overexpression of VQ8 or loss of WRKY10 function enhances thermotolerance. Overexpression of WRKY10 accelerates reactive oxygen species (ROS) accumulation in chloroplasts and apoplasts, and it also induces the expression of heat shock TF and protein genes. We also found that WRKY10 regulates nuclear DNA fragmentation and hypersensitive response by modulating NAC4 TF expression. The balance between destructive and protective responses in WRKY10-overexpression plant is more fragile and more easily broken by heat stress compared with wild type. In vitro and in vivo assays revealed that VQ8 interacts with WRKY10 and inhibits the transcription activity via repressing its DNA-binding activity. Our study demonstrates that WRKY10 negatively regulates thermotolerance by modulating the ROS balance and the hypersensitive response and that VQ8 functions antagonistically to positively regulate thermotolerance. The functional module of WRKY10-VQ8 provides safe and effective regulatory mechanisms in the heat stress response., (© 2022 John Wiley & Sons Ltd.)

Many transcription factors (TFs) in animals bind to both DNA and mRNA, regulating transcription and mRNA turnover. However, whether plant TFs function at both the transcriptional and post-transcriptional levels remains unknown. The rice (Oryza sativa) bZIP TF AVRPIZ-T-INTERACTING PROTEIN 5 (APIP5) negatively regulates programmed cell death and blast resistance and is targeted by the effector AvrPiz-t of the blast fungus Magnaporthe oryzae. We demonstrate that the nuclear localization signal of APIP5 is essential for APIP5-mediated suppression of cell death and blast resistance. APIP5 directly targets two genes that positively regulate blast resistance: the cell wall-associated kinase gene OsWAK5 and the cytochrome P450 gene CYP72A1. APIP5 inhibits OsWAK5 expression and thus limits lignin accumulation; moreover, APIP5 inhibits CYP72A1 expression and thus limits reactive oxygen species production and defense compounds accumulation. Remarkably, APIP5 acts as an RNA-binding protein to regulate mRNA turnover of the cell death- and defense-related genes OsLSD1 and OsRac1. Therefore, APIP5 plays dual roles, acting as TF to regulate gene expression in the nucleus and as an RNA-binding protein to regulate mRNA turnover in the cytoplasm, a previously unidentified regulatory mechanism of plant TFs at the transcriptional and post-transcriptional levels., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Dysfunction of susceptibility genes leads to broad-spectrum resistance (BSR) in plants. A rice ubiquitin E3 ligase degrades an ortholog of plant thermosensor to positively regulate blast resistance. Gao et al. recently showed that this E3 ligase also ubiquitinates a Ca 2+ -sensor to mediate rice BSR, extending our knowledge of susceptibility protein ubiquitination for plant BSR., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Phenolamides (PAs), a diverse group of specialized metabolites, including hydroxycinnamoylputrescine (HP), hydroxycinnamoylagmatine, and hydroxycinnamoyltryptamine, are important in plant resistance to biotic stress. However, the genes involved in the biosynthesis and modulation of PAs have not been fully elucidated. This study identified an HP biosynthetic gene cluster in rice (Oryza sativa) comprising one gene (OsODC) encoding a decarboxylase and two tandem-duplicated genes (OsPHT3 and OsPHT4) encoding putrescine hydroxycinnamoyl acyltransferases coexpressed in different tissues. OsODC catalyzes the conversion of ornithine to putrescine, which is used in HP biosynthesis involving OsPHT3 and OsPHT4. OsPHT3 or OsPHT4 overexpression causes HP accumulation and cell death and putrescine hydroxycinnamoyl acyltransferases (PHT) activity-dependent resistance against the fungal pathogen Magnaporthe oryzae. OsODC overexpression plants also confer enhanced resistance to M. oryzae. Notably, the basic leucine zipper transcription factor APIP5, a negative regulator of cell death, directly binds to the OsPHT4 promoter, repressing its transcription. Moreover, APIP5 suppression induces OsPHT4 expression and HP accumulation. Comparative genomic analysis revealed that the HP biosynthetic gene cluster is conserved in monocots. These results characterized a previously unidentified monocot-specific gene cluster that is involved in HP biosynthesis and contributes to defense and cell death in rice., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2021 Science China Press. Published by Elsevier B.V. All rights reserved.)

Genomic clustering of non-homologous genes for the biosynthesis of plant defensive compounds is an emerging theme, but insights into their formation and physiological function remain limited. Here we report the identification of a newly discovered hydroxycinnamoyl tyramine (HT) gene cluster in rice. This cluster contains a pyridoxamine 5'-phosphate oxidase (OsPDX3) producing the cofactor pyridoxal 5'-phosphate (PLP), a PLP-dependent tyrosine decarboxylase (OsTyDC1), and two duplicated hydroxycinnamoyl transferases (OsTHT1 and OsTHT2). These members were combined to represent an enzymological innovation gene cluster. Natural variation analysis showed that the abundance of the toxic tyramine intermediate of the gene cluster among different rice accessions is mainly determined by the coordinated transcription of OsTyDC1 and OsTHT1. Further pathogen incubation assays demonstrated that the end products of the HT gene cluster displayed enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. Oryzae (Xoo) and fungal pathogen Magnaporthe oryzae (M. oryzae), and the enhanced resistance is associated with the boost of phytoalexins and the activation of defense response. The unique presence of the HT gene cluster in Oryza AA genome, together with the enrichment of transposon elements within this gene cluster region, provides an evolutionary background to accelerate cluster member combinations. Our study not only discovered a gene cluster involved in the phenylpropanoid metabolism but also addressed the key aspects of gene cluster formation. In addition, our results provide a new metabolic pool for plant defense against pathogens., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2021 Science China Press. Published by Elsevier B.V. All rights reserved.)

Upon fungal and bacterial pathogen attack, plants launch pattern-triggered immunity (PTI) by recognizing pathogen-associated molecular patterns (PAMPs) to defend against pathogens. Although PTI-mediated response has been widely studied, a systematic understanding of the reprogrammed cellular processes during PTI by multi-omics analysis is lacking. In this study, we generated metabolome, transcriptome, proteome, ubiquitome and acetylome data to investigate rice (Oryza sativa) PTI responses to two PAMPs, the fungi-derived chitin and the bacteria-derived flg22. Integrative multi-omics analysis uncovered convergence and divergence of rice responses to these PAMPs at multiple regulatory layers. Rice responded to chitin and flg22 in a similar manner at the transcriptome and proteome levels, but distinct at the metabolome level. We found that this was probably due to post-translational regulation including ubiquitination and acetylation, which reshaped gene expression by modulating enzymatic activities, and possibly led to distinct metabolite profiles. We constructed regulatory atlas of metabolic pathways, including the defence-related phenylpropanoid and flavonoid biosynthesis and linoleic acid derivative metabolism. The multi-level regulatory network generated in this study sets the foundation for in-depth mechanistic dissection of PTI in rice and potentially in other related poaceous crop species., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Plant immunity is activated upon pathogen perception and often affects growth and yield when it is constitutively active. How plants fine-tune immune homeostasis in their natural habitats remains elusive. Here, we discover a conserved immune suppression network in cereals that orchestrates immune homeostasis, centering on a Ca 2+ -sensor, RESISTANCE OF RICE TO DISEASES1 (ROD1). ROD1 promotes reactive oxygen species (ROS) scavenging by stimulating catalase activity, and its protein stability is regulated by ubiquitination. ROD1 disruption confers resistance to multiple pathogens, whereas a natural ROD1 allele prevalent in indica rice with agroecology-specific distribution enhances resistance without yield penalty. The fungal effector AvrPiz-t structurally mimics ROD1 and activates the same ROS-scavenging cascade to suppress host immunity and promote virulence. We thus reveal a molecular framework adopted by both host and pathogen that integrates Ca 2+ sensing and ROS homeostasis to suppress plant immunity, suggesting a principle for breeding disease-resistant, high-yield crops., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Rice is a key food security crop in Africa. The importance of rice has led to increasing country-specific, regional, and multinational efforts to develop germplasm and policy initiatives to boost production for a more food-secure continent. Currently, this critically important cereal crop is predominantly cultivated by small-scale farmers under suboptimal conditions in most parts of sub-Saharan Africa (SSA). Rice blast disease, caused by the fungus Magnaporthe oryzae , represents one of the major biotic constraints to rice production under small-scale farming systems of Africa, and developing durable disease resistance is therefore of critical importance. In this review, we provide an overview of the major advances by a multinational collaborative research effort to enhance sustainable rice production across SSA and how it is affected by advances in regional policy. As part of the multinational effort, we highlight the importance of joint international partnerships in tackling multiple crop production constraints through integrated research and outreach programs. More specifically, we highlight recent progress in establishing international networks for rice blast disease surveillance, farmer engagement, monitoring pathogen virulence spectra, and the establishment of regionally based blast resistance breeding programs. To develop blast-resistant, high yielding rice varieties for Africa, we have established a breeding pipeline that utilizes real-time data of pathogen diversity and virulence spectra, to identify major and minor blast resistance genes for introgression into locally adapted rice cultivars. In addition, the project has developed a package to support sustainable rice production through regular stakeholder engagement, training of agricultural extension officers, and establishment of plant clinics.

RNase P functions either as a catalytic ribonucleoprotein (RNP) or as an RNA-free polypeptide to catalyse RNA processing, primarily tRNA 5' maturation. To the growing evidence of non-canonical roles for RNase P RNP subunits including regulation of chromatin structure and function, we add here a role for the rice RNase P Rpp30 in innate immunity. This protein (encoded by LOC_Os11g01074) was uncovered as the top hit in yeast two-hybrid assays performed with the rice histone deacetylase HDT701 as bait. We showed that HDT701 and OsRpp30 are localized to the rice nucleus, OsRpp30 expression increased post-infection by Pyricularia oryzae (syn. Magnaporthe oryzae), and OsRpp30 deacetylation coincided with HDT701 overexpression in vivo. Overexpression of OsRpp30 in transgenic rice increased expression of defence genes and generation of reactive oxygen species after pathogen-associated molecular pattern elicitor treatment, outcomes that culminated in resistance to a fungal (P. oryzae) and a bacterial (Xanthomonas oryzae pv. oryzae) pathogen. Knockout of OsRpp30 yielded the opposite phenotypes. Moreover, HA-tagged OsRpp30 co-purified with RNase P pre-tRNA cleavage activity. Interestingly, OsRpp30 is conserved in grass crops, including a near-identical C-terminal tail that is essential for HDT701 binding and defence regulation. Overall, our results suggest that OsRpp30 plays an important role in rice immune response to pathogens and provides a new approach to generate broad-spectrum disease-resistant rice cultivars., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Rice blast and bacterial blight are important diseases of rice (Oryza sativa) caused by the fungus Magnaporthe oryzae and the bacterium Xanthomonas oryzae pv. oryzae (Xoo), respectively. Breeding rice varieties for broad-spectrum resistance is considered the most effective and sustainable approach to controlling both diseases. Although dominant resistance genes have been extensively used in rice breeding and production, generating disease-resistant varieties by altering susceptibility (S) genes that facilitate pathogen compatibility remains unexplored. Here, using CRISPR/Cas9 technology, we generated loss-of-function mutants of the S genes Pi21 and Bsr-d1 and showed that they had increased resistance to M. oryzae. We also generated a knockout mutant of the S gene Xa5 that showed increased resistance to Xoo. Remarkably, a triple mutant of all three S genes had significantly enhanced resistance to both M. oryzae and Xoo. Moreover, the triple mutant was comparable to the wild type in regard to key agronomic traits, including plant height, effective panicle number per plant, grain number per panicle, seed setting rate, and thousand-grain weight. These results demonstrate that the simultaneous editing of multiple S genes is a powerful strategy for generating new rice varieties with broad-spectrum resistance., (© 2021 Institute of Botany, Chinese Academy of Sciences.)

Background: Plant pathogenic isolates of Rhizoctonia solani anastomosis group 1-intraspecific group IA (AG1-IA) infect a wide range of crops causing diseases such as rice sheath blight (ShB). ShB has become a serious disease in rice production worldwide. Additional genome sequences of the rice-infecting R. solani isolates from different geographical regions will facilitate the identification of important pathogenicity-related genes in the fungus., Results: Rice-infecting R. solani isolates B2 (USA), ADB (India), WGL (India), and YN-7 (China) were selected for whole-genome sequencing. Single-Molecule Real-Time (SMRT) and Illumina sequencing were used for de novo sequencing of the B2 genome. The genomes of the other three isolates were then sequenced with Illumina technology and assembled using the B2 genome as a reference. The four genomes ranged from 38.9 to 45.0 Mbp in size, contained 9715 to 11,505 protein-coding genes, and shared 5812 conserved orthogroups. The proportion of transposable elements (TEs) and average length of TE sequences in the B2 genome was nearly 3 times and 2 times greater, respectively, than those of ADB, WGL and YN-7. Although 818 to 888 putative secreted proteins were identified in the four isolates, only 30% of them were predicted to be small secreted proteins, which is a smaller proportion than what is usually found in the genomes of cereal necrotrophic fungi. Despite a lack of putative secondary metabolite biosynthesis gene clusters, the rice-infecting R. solani genomes were predicted to contain the most carbohydrate-active enzyme (CAZyme) genes among all 27 fungal genomes used in the comparative analysis. Specifically, extensive enrichment of pectin/homogalacturonan modification genes were found in all four rice-infecting R. solani genomes., Conclusion: Four R. solani genomes were sequenced, annotated, and compared to other fungal genomes to identify distinctive genomic features that may contribute to the pathogenicity of rice-infecting R. solani. Our analyses provided evidence that genomic conservation of R. solani genomes among neighboring AGs was more diversified than among AG1-IA isolates and the presence of numerous predicted pectin modification genes in the rice-infecting R. solani genomes that may contribute to the wide host range and virulence of this necrotrophic fungal pathogen.

Nucleotide-binding leucine-rich repeat (NLR) proteins play critical roles in plant immunity. However, how NLRs are regulated and activate defense signaling is not fully understood. The rice (Oryza sativa) NLR receptor Piz-t confers broad-spectrum resistance to the fungal pathogen Magnaporthe oryzae and the RING-type E3 ligase AVRPIZ-T INTERACTING PROTEIN 10 (APIP10) negatively regulates Piz-t accumulation. In this study, we found that APIP10 interacts with two rice transcription factors, VASCULAR PLANT ONE-ZINC FINGER 1 (OsVOZ1) and OsVOZ2, and promotes their degradation through the 26S proteasome pathway. OsVOZ1 displays transcriptional repression activity while OsVOZ2 confers transcriptional activation activity in planta. The osvoz1 and osvoz2 single mutants display modest but opposite M. oryzae resistance in the non-Piz-t background. However, the osvoz1 osvoz2 double mutant exhibits strong dwarfism and cell death, and silencing of both genes via RNA interference also leads to dwarfism, mild cell death, and enhanced resistance to M. oryzae in the non-Piz-t background. Both OsVOZ1 and OsVOZ2 interact with Piz-t. Double silencing of OsVOZ1 and OsVOZ2 in the Piz-t background decreases Piz-t protein accumulation and transcription, reactive oxygen species-dependent cell death, and resistance to M. oryzae containing AvrPiz-t. Taken together, these results indicate that OsVOZ1 and OsVOZ2 negatively regulate basal defense but contribute positively to Piz-t-mediated immunity., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The rice black-streaked dwarf virus (RBSDV) disease causes severe rice yield losses in Asia. RNA interference (RNAi) has been widely applied to develop antiviral varieties in plants. So far, only a few studies reported the application of RNAi in rice against RBSDV and most of them are lack of enough data to support its breeding potential, which limited the progress on developing RBSDV-resistant variety. In this study, we generated three RNAi constructs to specifically target three RBSDV genes (S1, S2 and S6), respectively. We confirmed that RNAi targeting RBSDV S6 conferred rice with almost full immunity to RBSDV through phenotyping test in eight consecutive years in both artificial inoculation and field trials, while RNAi of S1 or S2 only leads to partially increased resistance. The S6RNAi was also found conferring strong resistance to southern rice black-streaked dwarf virus (SRBSDV), a novel species closely related to RBSDV that outbroke recently in Southern China. In particular, no adverse effects on agronomical and developmental traits were found in S6RNAi transgenic lines. The marker-free transgenic lines with S6RNAi, driven by either maize ubiquitin-1 promoter or rice rbcS green tissue expression promoter, in elite rice background should have great potential in breeding of resistant varieties to both RBSDV and SRBSDV and provide a basis for further safety evaluation and commercial application., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Transcriptional re-programming in host and pathogen upon leaf and neck infection is an evolving area of research for the rice blast community. Analysis of in planta rice transcriptome in leaf and neck tissues revealed tissue-specific and infection-specific expression of rice and Magnaporthe oryzae genes in host and pathogen. The glycosyl hydrolase, isocitrate lyase, cupin domain containing protein, TF2, CMPG1, CHIT17 and OsCML14 genes were uniquely expressed in leaf infection. Genes like cytochrome P450, inhibitor I family protein, GSTU6, abscisic stress ripening, and cupin domain containing protein were up-regulated during neck infection. In our microRNA sequencing study, Osa-miR166n-3p was highly expressed in upon Magnaporthe leaf infection, whereas osa-miR1661-3p, osa-miR166n-3p and osa-miR159b were overexpressed in neck infection. Here we report several transcripts being targeted by up and down regulated microRNAs during infection. The putative genes expressed upon infection in leaf and neck could be used in understanding the dual-epidemics of blast disease., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mitochondria are essential for animal and plant immunity. Here, we report that the effector MoCDIP4 of the fungal pathogen Magnaporthe oryzae targets the mitochondria-associated OsDjA9-OsDRP1E protein complex to reduce rice immunity. The DnaJ protein OsDjA9 interacts with the dynamin-related protein OsDRP1E and promotes the degradation of OsDRP1E, which functions in mitochondrial fission. By contrast, MoCDIP4 binds OsDjA9 to compete with OsDRP1E, resulting in OsDRP1E accumulation. Knockout of OsDjA9 or overexpression of OsDRP1E or MoCDIP4 in transgenic rice results in shortened mitochondria and enhanced susceptibility to M. oryzae Overexpression of OsDjA9 or knockout of OsDRP1E in transgenic rice, in contrast, leads to elongated mitochondria and enhanced resistance to M. oryzae Our study therefore reveals a previously unidentified pathogen-infection strategy in which the pathogen delivers an effector into plant cells to target an HSP40-DRP complex; the targeting leads to the perturbation of mitochondrial dynamics, thereby inhibiting mitochondria-mediated plant immunity., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Bowman-Birk trypsin inhibitors (BBIs) play important roles in animal and plant immunity, but how these protease inhibitors are involved in the immune system remains unclear. Here, we show that the rice (Oryza sativa) BBI protein APIP4 is a common target of a fungal effector and an NLR receptor for innate immunity. APIP4 exhibited trypsin inhibitor activity in vitro and in vivo. Knockout of APIP4 in rice enhanced susceptibility, and overexpression of APIP4 increased resistance to the fungal pathogen Magnaporthe oryzae. The M. oryzae effector AvrPiz-t interacted with APIP4 and suppressed APIP4 trypsin inhibitor activity. By contrast, the rice NLR protein Piz-t interacted with APIP4, enhancing APIP4 transcript and protein levels, and protease inhibitor activity. Our findings reveal a novel host defence mechanism in which a host protease inhibitor targeted by a fungal pathogen is protected by an NLR receptor., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Because of the frequent breakdown of major resistance (R) genes, identification of new partial R genes against rice blast disease is an important goal of rice breeding. In this study, we used a core collection of the Rice Diversity Panel II (C-RDP-II), which contains 584 rice accessions and are genotyped with 700 000 single-nucleotide polymorphism (SNP) markers. The C-RDP-II accessions were inoculated with three blast strains collected from different rice-growing regions in China. Genome-wide association study identified 27 loci associated with rice blast resistance (LABRs). Among them, 22 LABRs were not associated with any known blast R genes or QTLs. Interestingly, a nucleotide-binding site leucine-rich repeat (NLR) gene cluster exists in the LABR12 region on chromosome 4. One of the NLR genes is highly conserved in multiple partially resistant rice cultivars, and its expression is significantly up-regulated at the early stages of rice blast infection. Knockout of this gene via CRISPR-Cas9 in transgenic plants partially reduced blast resistance to four blast strains. The identification of this new non-strain specific partial R gene, tentatively named rice blast Partial Resistance gene 1 (PiPR1), provides genetic material that will be useful for understanding the partial resistance mechanism and for breeding durably resistant cultivars against blast disease of rice., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Protein arginine methyltransferases (PRMTs) regulate many physiological processes, including autophagy. However, the direct roles of the various PRMTs during autophagosome formation remain unclear. Here, we characterised the function of MoHMT1 in the rice blast fungus, Magnaporthe oryzae. Knockout of MoHMT1 results in inhibited growth and a decreased ability to cause disease lesions on rice seedlings. MoHMT1 catalyses the di-methylation of arginine 247, 251, 261 and 271 residues of MoSNP1, a U1 small nuclear ribonucleoprotein (snRNP) component, likely in a manner dependent on direct interaction. RNA-seq analysis revealed that alternative splicing of pre-mRNAs of 558 genes, including the autophagy-related (ATG) gene MoATG4, was altered in MoHMT1 deletion mutants, compared with wild-type strains under normal growth conditions. During light exposure or nitrogen starvation, MoHMT1 localises to autophagosomes and MoHMT1 mutants display defects in autophagy induction. Under nitrogen starvation, six additional MoATG genes were identified with retained introns in their mRNA transcripts, corresponding with a significant reduction in transcripts of intron-spliced isoforms in the MoHMT1 mutant strain. Our study shows that arginine methylation plays an essential role in accurate pre-mRNA splicing necessary for a range of developmental processes, including autophagosome formation., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Lysine succinylation (Ksu) is a dynamic and reversible post-translational modification that plays an important role in many biological processes. Although recent research has analyzed Ksu plant proteomes, little is known about the scope and cellular distribution of Ksu in rice seedlings. Here, we report high-quality proteome-scale Ksu data for rice seedlings. A total of 710 Ksu sites in 346 proteins with diverse biological functions and subcellular localizations were identified in rice samples. About 54% of the sites were predicted to be localized in the chloroplast. Six putative succinylation motifs were detected. Comparative analysis with succinylation data revealed that arginine (R), located downstream of Ksu sites, is the most conserved amino acid surrounding the succinylated lysine. KEGG pathway category enrichment analysis indicated that carbon metabolism, tricarboxylic acid cycle (TCA) cycle, oxidative phosphorylation, photosynthesis, and glyoxylate and dicarboxylate metabolism pathways were significantly enriched. Additionally, we compared published Ksu data from rice embryos with our data from rice seedlings and found conserved Ksu sites between the two rice tissues. Our in-depth survey of Ksu in rice seedlings provides the foundation for further understanding the biological function of lysine-succinylated proteins in rice growth and development.

Lesion mimic mutants are ideal genetic materials to study programmed cell death and defense signaling in plants. However, the molecular basis of lesion mimic formation remains largely unknown. Here, we first used a proteomic approach to identify differentially expressed proteins during dynamic lesion mimic formation in the rice oscul3a mutant, then electron microscope observation and physiological assays were used to analyze the mutant. The oscul3a mutant had disrupted cell metabolism balance, and the identified differentially expressed proteins were mainly located in the chloroplast and cytoplasm, which caused enhanced lipid metabolism, but suppressed carbon/nitrogen metabolism with reduced growth and grain quality. The oscul3a mutant had higher salicylic acid (SA) concentration in leaves, and H 2 O 2 was shown to accumulate late in the formation of lesions. The secondary metabolite coumarin induced reactive oxygen species (ROS) and had rice blast resistance activity. Moreover, the cell death initiated lesion mimic formation of oscul3a mutant was light-sensitive, which might be associated with metabolite biosynthesis and accumulation. This study sheds light on the metabolic transition associated with cell death and defense response, which is under tight regulation by OsCUL3a and metabolism-related proteins, and the newly identified chemicals in the secondary metabolic pathway can potentially be used to control disease in crop plants., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Phenylalanine ammonia lyases (PALs) are involved in resistance to plant pathogens, but the mechanism by which they contribute to plant immunity is unclear. Two recent studies provide direct evidence for PALs' contributions to rice broad-spectrum resistance (BSR) mediated by nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins and RNA-binding proteins (Zhai et al., Mol. Cell, 2019; Zhou et al., Proc. Natl. Acad. Sci. U. S. A., 2018)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Environmental conditions are key factors in the progression of plant disease epidemics. Light affects the outbreak of plant diseases, but the underlying molecular mechanisms are not well understood. Here, we report that the light-harvesting complex II protein, LHCB5, from rice is subject to light-induced phosphorylation during infection by the rice blast fungus Magnaporthe oryzae We demonstrate that single-nucleotide polymorphisms (SNPs) in the LHCB5 promoter control the expression of LHCB5 , which in turn correlates with the phosphorylation of LHCB5. LHCB5 phosphorylation enhances broad-spectrum resistance of rice to M. oryzae through the accumulation of reactive oxidative species (ROS) in the chloroplast. We also show that LHCB5 phosphorylation-induced resistance is inheritable. Our results uncover an immunity mechanism mediated by phosphorylation of light-harvesting complex II., Competing Interests: The authors declare no conflict of interest.

Magnaporthe oryzae is an important fungal pathogen of both rice and wheat. However, how M. oryzae effectors modulate plant immunity is not fully understood. Previous studies have shown that the M. oryzae effector AvrPiz-t targets the host ubiquitin-proteasome system to manipulate plant defence. In return, two rice ubiquitin E3 ligases, APIP6 and APIP10, ubiquitinate AvrPiz-t for degradation. To determine how lysine residues contribute to the stability and function of AvrPiz-t, we generated double (K1,2R-AvrPiz-t), triple (K1,2,3R-AvrPiz-t) and lysine-free (LF-AvrPiz-t) mutants by mutating lysines into arginines in AvrPiz-t. LF-AvrPiz-t showed the highest protein accumulation when transiently expressed in rice protoplasts. When co-expressed with APIP10 in Nicotiana benthamiana, LF-AvrPiz-t was more stable than AvrPiz-t and was less able to degrade APIP10. The avirulence of LF-AvrPiz-t on Piz-t:HA plants was less than that of AvrPiz-t, which led to resistance reduction and lower accumulation of the Piz-t:HA protein after inoculation with the LF-AvrPiz-t-carrying isolate. Chitin- and flg22-induced production of reactive oxygen species (ROS) was higher in LF-AvrPiz-t than in AvrPiz-t transgenic plants. In addition, LF-AvrPiz-t transgenic plants were less susceptible than AvrPiz-t transgenic plants to a virulent isolate. Furthermore, both AvrPiz-t and LF-AvrPiz-t interacted with OsRac1, but the suppression of OsRac1-mediated ROS generation by LF-AvrPiz-t was significantly lower than that by AvrPiz-t. Together, these results suggest that the lysine residues of AvrPiz-t are required for its avirulence and virulence functions in rice., (© 2018 The Authors. Molecular Plant Pathology Published by BSPP and John Wiley & Sons Ltd.)

The absorption of nutrients and disease resistance are two indispensable physiological processes in plants; however, it is still largely unknown whether there is cross-talk between their molecular signaling pathways. In this study, we identified the rice OsPT8 protein, which is a member of the phosphate transporters (PTs) Pht1 family and also plays a role in rice disease resistance. The transcriptional level of OsPT8 is suppressed after infection with rice pathogens and treatment with pathogen-associated molecular patterns (PAMPs). Overexpression of OsPT8 suppresses rice disease resistance against the pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae. Accordingly, the transcription level of resistance related genes, such as PAL and PBZ1, is inhibited in plants overexpressing OsPT8 (OsPT8-OX) after inoculation with these pathogens. In OsPT8-OX plants, PAMPs-triggered immunity (PTI) response genes, such as OsRac1 and SGT1, are suppressed during treatment with PAMPs chitin or flg22. Moreover, the typical response of PTI is suppressed after chitin or flg22 treatment. We also identified OsPT8 as an interactor of a rice mitogen-activated protein kinase BWMK1, which is a regulator of disease resistance. Under low phosphate (Pi) conditions, the OsPT8-OX plants display better agronomic traits than the control plants. However, the differences in development between OsPT8-OX and the control plants are reduced upon the increase of Pi concentration. These results demonstrate that OsPT8 regulates the transduction of Pi signaling for development and negatively regulates rice immunity.

Plant resistance genes typically encode proteins with nucleotide binding site-leucine rich repeat (NLR) domains. Here we show that Ptr is an atypical resistance gene encoding a protein with four Armadillo repeats. Ptr is required for broad-spectrum blast resistance mediated by the NLR R gene Pi-ta and by the associated R gene Pi-ta2. Ptr is expressed constitutively and encodes two isoforms that are mainly localized in the cytoplasm. A two base pair deletion within the Ptr coding region in the fast neutron-generated mutant line M2354 creates a truncated protein, resulting in susceptibility to M. oryzae. Targeted mutation of Ptr in a resistant cultivar using CRISPR/Cas9 leads to blast susceptibility, further confirming its resistance function. The cloning of Ptr may aid in the development of broad spectrum blast resistant rice.

Programmed cell death (PCD) plays critical roles in plant immunity but must be regulated to prevent excessive damage. The E3 ubiquitin ligase SPL11 negatively regulates PCD and immunity in plants. We show that SPL11 cell-death suppressor 2 (SDS2), an S-domain receptor-like kinase, positively regulates PCD and immunity in rice by engaging and regulating SPL11 and related kinases controlling defense responses. An sds2 mutant shows reduced immune responses and enhanced susceptibility to the blast fungus Magnaporthe oryzae. Conversely, SDS2 over-expression induces constitutive PCD accompanied by elevated immune responses and enhanced resistance to M. oryzae. SDS2 interacts with and phosphorylates SPL11, which in turn ubiquitinates SDS2, leading to its degradation. In addition, SDS2 interacts with related receptor-like cytoplasmic kinases, OsRLCK118/176, that positively regulate immunity by phosphorylating the NADPH oxidase OsRbohB to stimulate ROS production. Thus, a plasma membrane-resident protein complex consisting of SDS2, SPL11, and OsRLCK118/176 controls PCD and immunity in rice., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Rice blast, caused by Magnaporthe oryzae (synonym: Pyricularia oryzae), severely reduces rice production and grain quality. The molecular mechanism of rice resistance to M. oryzae is not fully understood. In this study, we identified a chaperone DnaJ protein, OsDjA6, which is involved in basal resistance to M. oryzae in rice. The OsDjA6 protein is distributed in the entire rice cell. The expression of OsDjA6 is significantly induced in rice after infection with a compatible isolate. Silencing of OsDjA6 in transgenic rice enhances resistance to M. oryzae and also results in an increased burst of reactive oxygen species after flg22 and chitin treatments. In addition, the expression levels of WRKY45, NPR1 and PR5 are increased in OsDjA6 RNAi plants, indicating that OsDjA6 may mediate resistance by affecting the salicylic acid pathway. Finally, we found that OsDjA6 interacts directly with the E3 ligase OsZFP1 in vitro and in vivo. These results suggest that the DnaJ protein OsDjA6 negatively regulates rice innate immunity, probably via the ubiquitination proteasome degradation pathway., (© 2017 BSPP AND JOHN WILEY & SONS LTD.)

Potassium (K+) is required by plants for growth and development, and also contributes to immunity against pathogens. However, it has not been established whether pathogens modulate host K+ signaling pathways to enhance virulence and subvert host immunity. Here, we show that the effector protein AvrPiz-t from the rice blast pathogen Magnaporthe oryzae targets a K+ channel to subvert plant immunity. AvrPiz-t interacts with the rice plasma-membrane-localized K+ channel protein OsAKT1 and specifically suppresses the OsAKT1-mediated K+ currents. Genetic and phenotypic analyses show that loss of OsAKT1 leads to decreased K+ content and reduced resistance against M. oryzae. Strikingly, AvrPiz-t interferes with the association of OsAKT1 with its upstream regulator, the cytoplasmic kinase OsCIPK23, which also plays a positive role in K+ absorption and resistance to M. oryzae. Furthermore, we show a direct correlation between blast disease resistance and external K+ status in rice plants. Together, our data present a novel mechanism by which a pathogen suppresses plant host immunity by modulating a host K+ channel.

Understanding the genetic diversity of rice germplasm is important for the sustainable use of genetic materials in rice breeding and production. Africa is rich in rice genetic resources that can be utilized to boost rice productivity on the continent. A major constraint to rice production in Africa is rice blast, caused by the hemibiotrophic fungal pathogen Magnaporthe oryzae. In this report, we present the results of a genotyping-by-sequencing (GBS)-based diversity analysis of 190 African rice cultivars and an association mapping of blast resistance (R) genes and quantitative trait loci (QTLs). The 190 African cultivars were clustered into three groups based on the 184K single nucleotide polymorphisms generated by GBS. We inoculated the rice cultivars with six African M. oryzae isolates. Association mapping identified 25 genomic regions associated with blast resistance (RABRs) in the rice genome. Moreover, PCR analysis indicated that RABR_23 is associated with the Pi-ta gene on chromosome 12. Our study demonstrates that the combination of GBS-based genetic diversity population analysis and association mapping is effective in identifying rice blast R genes/QTLs that contribute to resistance against African populations of M. oryzae. The identified markers linked to the RABRs and 14 highly resistant cultivars in this study will be useful for rice breeding in Africa.

Avirulence (AVR) genes in Magnaporthe oryzae, the fungal pathogen that causes the devastating rice blast disease, have been documented to be major targets subject to mutations to avoid recognition by resistance (R) genes. In this study, an AVR-gene-based diagnosis tool for determining the virulence spectrum of a rice blast pathogen population was developed and validated. A set of 77 single-spore field isolates was subjected to pathotype analysis using differential lines, each containing a single R gene, and classified into 20 virulent pathotypes, except for 4 isolates that lost pathogenicity. In all, 10 differential lines showed low frequency (<24%) of resistance whereas 8 lines showed a high frequency (>95%), inferring the effectiveness of R genes present in the respective differential lines. In addition, the haplotypes of seven AVR genes were determined by polymerase chain reaction amplification and sequencing, if applicable. The calculated frequency of different AVR genes displayed significant variations in the population. AVRPiz-t and AVR-Pii were detected in 100 and 84.9% of the isolates, respectively. Five AVR genes such as AVR-Pik-D (20.5%) and AVR-Pik-E (1.4%), AVRPiz-t (2.7%), AVR-Pita (0%), AVR-Pia (0%), and AVR1-CO39 (0%) displayed low or even zero frequency. The frequency of AVR genes correlated almost perfectly with the resistance frequency of the cognate R genes in differential lines, except for International Rice Research Institute-bred blast-resistant lines IRBLzt-T, IRBLta-K1, and IRBLkp-K60. Both genetic analysis and molecular marker validation revealed an additional R gene, most likely Pi19 or its allele, in these three differential lines. This can explain the spuriously higher resistance frequency of each target R gene based on conventional pathotyping. This study demonstrates that AVR-gene-based diagnosis provides a precise, R-gene-specific, and differential line-free assessment method that can be used for determining the virulence spectrum of a rice blast pathogen population and for predicting the effectiveness of target R genes in rice varieties.

Cullin3-based RING E3 ubiquitin ligases (CRL3), composed of Cullin3 (CUL3), RBX1, and BTB proteins, are involved in plant immunity, but the function of CUL3 in the process is largely unknown. Here, we show that rice ( Oryza sativa ) OsCUL3a is important for the regulation of cell death and immunity. The rice lesion mimic mutant oscul3a displays a significant increase in the accumulation of flg22- and chitin-induced reactive oxygen species, and in pathogenesis-related gene expression as well as resistance to Magnaporthe oryzae and Xanthomonas oryzae pv oryzae. We cloned the OsCUL3a gene via a map-based strategy and found that the lesion mimic phenotype of oscul3a is associated with the early termination of OsCUL3a protein. Interaction assays showed that OsCUL3a interacts with both OsRBX1a and OsRBX1b to form a multisubunit CRL in rice. Strikingly, OsCUL3a interacts with and degrades OsNPR1, which acts as a positive regulator of cell death in rice. Accumulation of OsNPR1 protein is greater in the oscul3a mutant than in the wild type. Furthermore, the oscul3a osnpr1 double mutant does not exhibit the lesion mimic phenotype of the oscul3a mutant. Our data demonstrate that OsCUL3a negatively regulates cell death and immunity by degrading OsNPR1 in rice., (© 2017 American Society of Plant Biologists. All rights reserved.)

Programmed cell death (PCD) mediated by mitochondrial processes has emerged as an important mechanism for plant development and responses to abiotic and biotic stresses. However, the role of translocation of cytochrome c from the mitochondria to the cytosol during PCD remains unclear. Here, we demonstrate that the rice dynamin-related protein 1E (OsDRP1E) negatively regulates PCD by controlling mitochondrial structure and cytochrome c release. We used a map-based cloning strategy to isolate OsDRP1E from the lesion mimic mutant dj-lm and confirmed that the E409V mutation in OsDRP1E causes spontaneous cell death in rice. Pathogen inoculation showed that dj-lm significantly enhances resistance to fungal and bacterial pathogens. Functional analysis of the E409V mutation showed that the mutant protein impairs OsDRP1E self-association and formation of a higher-order complex; this in turn reduces the GTPase activity of OsDRP1E. Furthermore, confocal microscopy showed that the E409V mutation impairs localization of OsDRP1E to the mitochondria. The E409V mutation significantly affects the morphogenesis of cristae in mitochondria and causes the abnormal release of cytochrome c from mitochondria into cytoplasm. Taken together, our results demonstrate that the mitochondria-localized protein OsDRP1E functions as a negative regulator of cytochrome c release and PCD in plants., Competing Interests: The authors have declared that no competing interests exist.