Nuclear processes underpin host phenotypic plasticity and environmental adaptation, but how distinct nuclear processes are coordinated in response to external biotic stimuli remains poorly understood. Here, we performed a genome-wide association study and identified OsTOPBP1C, which encodes a multifunctional protein that modulates rice resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae. We demonstrated that the Xoo-secreted transcription activator-like effector TalDR22GIV subverts host immunity by commandeering the transcriptional control of OsTOPBP1C. TalDR22GIV antagonizes OsTOPBP1C transcription induced by the avirulence protein TalAE73GIV and the DNA damage response integrator OsSOG1, thereby sustaining the DNA repair function of OsTOPBP1C and counteracting immune responses. Promoter activity assays and transcriptomic analyses revealed that OsTOPBP1C fine-tunes rice immunity through a transcription-dependent mechanism. Mechanistically, OsTOPBP1C plays two roles in rice immunity: during infection, reduced transcription shifts its function toward DNA repair, thereby promoting OsSOG1-mediated susceptibility; conversely, high levels of OsTOPBP1C transcription enable it to potentiate salicylic acid-mediated defenses by suppressing the transcription of OsSAH3 activated by OsMYC2. Our findings reveal a pathogen strategy for rewiring host nuclear processes and demonstrate how host gene transcription shapes immune plasticity, providing insight into the molecular choreography of host-pathogen interactions.

Rice is exposed to attacks by the three most destructive pathogens, Magnaporthe oryzae (M. oryzae), Xanthomonas oryzae pv. oryzae (Xoo), and Rhizoctonia solani (R. solani), which cause substantial yield losses and severely threaten food security. To cope with pathogenic infections, rice has evolved diverse molecular mechanisms to respond to a wide range of pathogens. Among these strategies, plant microRNAs (miRNAs), endogenous single-stranded short non-coding RNA molecules, have emerged as promising candidates in coordinating plant–pathogen interactions. MiRNAs can modulate target gene expression at the post-transcriptional level through mRNA cleavage and/or translational inhibition. In rare instances, they also influence gene expression at the transcriptional level through DNA methylation. In recent years, substantial advancements have been achieved in the investigation of microRNA-mediated molecular mechanisms in rice immunity. Therefore, we attempt to summarize the current advances of immune signaling mechanisms in rice–pathogen interactions that are regulated by osa-miRNAs, including their functions and molecular mechanisms. We also focus on recent findings concerning the role of osa-miRNAs that respond to M. oryzae, Xoo, and R. solani, respectively. These insights enhance our understanding of how the mechanisms of osa-miRNAs mediate rice immunity and may facilitate the development of improved strategies for breeding pathogen-resistant rice varieties.

Transcription activator-like effectors (TALEs) secreted from Xanthomonas oryzae pv oryzae (Xoo) function as a pathogenicity factor to activate rice bacterial blight (BB) susceptibility, conforming to the gene-for-gene paradigm as well as resistance. Xoo pathotypes generally harbor one to three major TALEs targeting OsSWEET genes to determine pathogenicity; conversely, the immunity events mediated by minor TALEs have not been taken seriously. Here, we demonstrated that lipid transfer protein encoding gene OsLTPL23 positively regulates rice resistance to Xoo pathotype PXO61, and TalAE73PXO61, a representative member of the most widely distributed TALE family in 135 Xoo isolates, transcriptionally activates OsLTPL23 expression. Further, TalAE73PXO61 is an avirulence protein, causing effector-triggered immunity in compatible rice-Xoo interaction. In addition, reactive oxygen species accumulation, nitrate uptake, and salicylic acid homeostasis are transcriptionally and physiologically associated with OsLTPL23-dependent BB resistance.

Xanthomonas oryzae pv. oryzae ( Xoo) is the causal bacterium of rice bacterial blight (BB), which is one of the most destructive bacterial diseases of rice worldwide. Although more than 40 BB resistance genes ( Xa genes) have been identified, only a few conferring broad resistance (e.g., Xa4 and Xa21) have been widely used for rice breeding in Asia. The narrow genetic basis for the BB resistance of rice cultivars has resulted in the emergence of new pathogenic strains that can overcome the resistance of commonly grown rice cultivars derived from breeding programs. For example, Xoo strain HN2011, which was isolated in Hainan (China), can overcome the resistance mediated by multiple BB resistance genes, including Xa4 and Xa21. We herein present the complete genome sequence of HN2011 generated by Nanopore sequencing. The genomic data will be useful for elucidating the genomic variations of Xoo and will contribute to clarifying the interaction between Xoo and rice. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Growing resistant rice cultivars is the most effective strategy to control bacterial blight (BB), a devastating disease caused by Xanthomonas oryzae pv. oryzae (Xoo). Screening resistant germplasm and identifying resistance (R) genes are prerequisites for breeding resistant rice cultivars. We conducted a genome-wide association study (GWAS) to detect quantitative trait loci (QTL) associated with BB resistance using 359 East Asian temperate Japonica accessions inoculated with two Chinese Xoo strains (KS6-6 and GV) and one Philippine Xoo strain (PXO99(A)). Based on the 55K SNPs Array dataset of the 359 Japonica accessions, eight QTL were identified on rice chromosomes 1, 2, 4, 10, and 11. Four of the QTL coincided with previously reported QTL, and four were novel loci. Six R genes were localized in the qBBV-11.1, qBBV-11.2, and qBBV-11.3 loci on chromosome 11 in this Japonica collection. Haplotype analysis revealed candidate genes associated with BB resistance in each QTL. Notably, LOC_Os11g47290 in qBBV-11.3, encoding a leucine-rich repeat receptor-like kinase, was a candidate gene associated with resistance to the virulent strain GV. Knockout mutants of Nipponbare with the susceptible haplotype of LOC_Os11g47290 exhibited significantly improved BB resistance. These results will be useful for cloning BB resistance genes and breeding resistant rice cultivars.

Seed germination is vital for ensuring the continuity of life in spermatophyte. High-quality seed germination usually represents good seedling establishment and plant production. Here, we identified OsLTPL23, a putative rice non-specific lipid transport protein, as an important regulator responsible for seed germination. Subcellular localization analysis confirmed that OsLTPL23 is present in the plasma membrane and nucleus. The knockout mutants of OsLTPL23 were generated by CRISPR/Cas9-mediated genome editing, and osltpl23 lines significantly germinated slower and lower than the Nipponbare (NIP). Starch and soluble sugar contents measurement showed that OsLTPL23 may have alpha-amylase inhibitor activity, and high soluble sugar content may be a causal agent for the delayed seed germination of osltpl23 mutants. Transcript profiles in the germinating seeds exhibited that the abscisic acid (ABA)-responsive genes, OsABI3 and OsABI5, and biosynthesis genes, OsNCED1, OsNCED2, OsNCED3 and OsNCED4, are obviously upregulated in the osltpl23 mutants compared to NIP plants, conversely, ABA metabolism genes OsABA8ox1, OsABA8ox2 and OsABA8ox3 are stepwise decreased. Further investigations found that osltpl23 mutants displays weakened early seedling growth, with elevated gene expresssion of ABA catabolism genes and repressive transcription response of defence-related genes OsWRKY45, OsEiN3, OsPR1a, OsPR1b and OsNPR1. Integrated analysis indicated that OsLTPL23 may exert an favorable effect on rice seed germination and early seedling growth via modulating endogenous ABA homeostasis. Collectively, our study provides important insights into the roles of OsLTPL23-mediated carbohydrate conversion and endogenous ABA pathway on seed germination and early seedling growth, which contributes to high-vigor seed production in rice breeding.

High yield and superior quality are the main goals pursued by breeders for crop improvement. However, both of them are complex agronomic traits controlled by multiple genes, so the simultaneous improvement of these traits via sexual recombination is time-consuming and direction-uncontrolled. In this study, to solve this dilemma, we introduced the comparative genomic analysis based multiplex genome editing system (CG-MGE), a method for rapid and directional improvement of multiple traits. Application of this method, association analysis between genotypes and phenotypes was carried out to mine excellent alleles; subsequently, the rare excellent alleles of Gn1a, GW2, TGW3, and Chalk5 were simultaneously created by multiplex genome editing and successfully improved the plant architecture, grain yield, and quality of a widely cultivated elite rice variety. Overall, this study provides a method for rapid and directional improvement of crops, and the application of the CG-MGE will be helpful to accelerate rational design breeding.

Papain-like cysteine proteases (PLCPs) play an important role in the immune response of plants. In Arabidopsis, several homologous genes are known to be involved in defending against pathogens. However, the effects of PLCPs on diseases that afflict rice are largely unknown. In this study, we show that a PLCP, an oryzain alpha chain precursor (OCP), the ortholog of the Arabidopsis protease RD21 (responsive to dehydration 21), participates in regulating resistance to blast disease with a shorter lesion length characterizing the knockout lines (ocp-ko), generated via CRISPR/Cas9 technology. OCP was expressed in all rice tissues and mainly located in the cytoplasm. We prove that OCP, featuring cysteine protease activity, interacts with OsRACK1A (receptor for activated C kinase 1) and OsSNAP32 (synaptosome-associated protein of 32 kD) physically in vitro and in vivo, and they co-locate in the rice cytoplasm but cannot form a ternary complex. Many genes related to plant immunity were enriched in the ocp-ko1 line whose expression levels changed significantly. The expression of jasmonic acid (JA) and ethylene (ET) biosynthesis and regulatory genes were up-regulated, while that of auxin efflux transporters was down-regulated in ocp-ko1. Therefore, OCP negatively regulates blast resistance in rice by interacting with OsRACK1A or OsSNAP32 and influencing the expression profiles of many resistance-related genes. Moreover, OCP might be the cornerstone of blast resistance by suppressing the activation of JA and ET signaling pathways as well as promoting auxin signaling pathways. Our research provides a comprehensive resource of PLCPs for rice plants in defense against pathogens that is also of potential breeding value.

Bacterial blight (BB), which is caused by Xanthomonas oryzae pv. oryzae (Xoo), is an important rice disease responsible for significant yield losses. In the rice-growing regions of South China where BB outbreaks are common, the resistance of cultivars with BB resistance genes Xa4 and Xa21 has been lost because of rapid changes in the Xoo population structure and virulence. In this study, 421 diverse rice accessions were evaluated regarding their resistance to two Xoo strains, namely GD1358 (C5) and IV, which are prevalent pathotypes in South China and overcame the resistance of Xa4 and Xa21, respectively. Using the 4.8mio filtered SNP dataset, we conducted a genome-wide association study, which identified 13 loci associated with BB resistance, including eight new quantitative trait loci (QTL) and five QTL harboring known BB resistance genes: Xa3/Xa26, xa5, Xa35(t), Xa36(t), Xa40, Xa43(t), and xa44(t). Intriguingly, a steep peak was detected on chromosomes 5 and 11. Six QTL including three new ones, were distributed on chromosome 11, whereas a new QTL qBB5.1 and a known QTL were detected on chromosome 5. Haplotype analyses indicated that the LOC_Os05g01610 (OsPRAF2) gene within the qBB5.1 region, which encodes a PRAF protein, is associated with BB resistance. Furthermore, OsPRAF2 knockout lines generated using the CRISPR-Cpf1 system were significantly more resistant to Xoo strains than the wild-type plants. Our results provide researchers and breeders with useful information regarding QTL and gene resources, which may be relevant for developing new BB-resistant rice cultivars.

The rapid increase of genetically modified organisms (GMOs) entering the food and feed markets, and the contamination of donor (micro)organisms of transgenic elements make it more challenging for the existing GMO detection. In this study, we developed a high-throughput and contamination-removal GMO detection approach named as GmoDetector. GmoDetector targeted 64 common transgenic elements and 76 GMO-specific events collected from 251 singular GM events, and combined with next generation sequencing (NGS) and target enrichment technology to detect various GMOs. As a result, GmoDetector was able to exclude the donor (micro)organism contamination, and detect the authorized and unauthorized GMOs (UGMOs) in any forms of food or feed, such as processed or unprocessed. The sensitivity of GmoDetector is as low as 0.1% (GMO content), which has met the GMO labeling threshold for all countries. Therefore, GmoDetector is a robust tool for accurate and efficient detection of the authorized and UGMOs.

MicroRNAs (miRNAs) handle immune response to pathogens by adjusting the function of target genes in plants. However, the experimentally documented miRNA/target modules implicated in the interplay between rice and Xanthomonas oryzae pv. oryzae (Xoo) are still in the early stages. Herein, the expression of osa-miR1432 was induced in resistant genotype IRBB5, but not susceptible genotype IR24, under Xoo strain PXO86 attack. Overexpressed osa-miR1432 heightened rice disease resistance to Xoo, indicated by enhancive enrichment of defense marker genes, raised reactive oxygen species (ROS) levels, repressed bacterial growth and shortened leaf lesion length, whilst the disruptive accumulation of osa-miR1432 accelerated rice susceptibility to Xoo infection. Noticeably, OsCaML2 (LOC_Os03g59770) was experimentally confirmed as a target gene of osa-miR1432, and the overexpressing OsCaML2 transgenic plants exhibited compromised resistance to Xoo infestation. Our results indicate that osa-miR1432 and OsCaML2 were differently responsive to Xoo invasion at the transcriptional level and fine-tune rice resistance to Xoo infection, which may be referable in resistance gene discovery and valuable in the pursuit of improving Xoo resistance in rice breeding.

The xa5 gene encodes a basal transcription factor (TFIIAγ) protein with wide spectrum resistance to bacterial blight caused by Xanthomonas oryzae pv. Oryzae (Xoo) in rice. It was only found in a few rice ecotypes, and the recessive characteristics limited its application in breeding. Here, we employed a TALEN-based technique to edit its dominant allelic TFIIAγ5 and obtained many mutant TFIIAγ5 genes. Most of them reduced rice susceptibility to varying degrees when the plants were challenged with the Xoo. In particular, the knocked-out TFIIAγ5 can reduce the rice susceptibility significantly, although it cannot reach the xa5-mediated resistance level, indicating TFIIAγ5 is a major component involved in disease susceptibility. In addition, the mutant encoding the protein with deletion of the 32nd amino acid or amino acid insertion between 32nd and 33rd site confers rice with the similar resistance to that of the knocked-out TFIIAγ5. Thus, the amino acids around 32nd site are also the important action sites of TFIIAγ5 besides the 39th amino acid previously reported. Moreover, the integration of xa5 into TFIIAγ5-knockout plants conferred them with a similar resistance as IRBB5, the rice variety containing the homozygous xa5 gene. Thus, TFIIAγ5 was not simply regarded as a resistant or a susceptible locus, as the substitution of amino acids might shift its functions.

MicroRNAs (miRNAs) are crucial player in plant-pathogen interaction. While the evidence has demonstrated that rice miRNAs mediate immune response to pathogens invasion, the roles of miRNAs on Xanthomonas oryzae pv. oryzae (Xoo) attack remain be in place. Herein, we monitored the responsive changes of rice miRNAs at 0, 8, 24 h across Xoo strain PXO86 infection in its compatible rice variety IR24 and incompatible variety IRBB5 by small RNA sequencing, and the genes targeted by miRNAs were also detected via degradome technology. The faithfulness of sequencing data was validated through quantitative real-time stem-loop reverse transcription-polymerase chain reaction assay. Bioinformatic analysis showed that the differentially expressed miRNAs could be divided into three immunity-related clusters, and 80 regulatory units were emerged in infection process, which comprises 29 differentially expressed known miRNAs and 38 cleaved targets. Furthermore, the miRNA presumptive function of separate immunity cluster in rice-Xoo interplay was confirmed through overexpressing osa-miR164a, osa-miR167d and osa-miR159b, and the disruption of regulatory units, osa-miR164a/OsNAC60, osa-miR167d-5p/OsWD40-174 and osa-miR159b/OsMYBGA, OsLRR-RLK2, OsMPK20-4, may reset rice defense response to Xoo infestation in a controllable manner. These findings provide new insights into the complex roles of characteristic miRNAs and their targets in rice-Xoo interactions.

Salicylic acid (SA) is a key natural component that mediates local and systemic resistance to pathogens in many dicotyledonous species. However, its function is controversial in disease resistance in rice plants. Here, we show that the SA signaling is involved in both pathogen-associated-molecular-patterns triggered immunity (PTI) and effector triggered immunity (ETI) to Xanthomonas oryzae pv. Oryzae (Xoo) mediated by the recessive gene xa5, in which OsNPR3.3 plays an important role through interacting with TGAL11. Rice plants containing homozygous xa5 gene respond positively to exogenous SA, and their endogenous SA levels are also especially induced upon infection by the Xoo strain, PXO86. Depletion of endogenous SA can significantly attenuate plant resistance to PXO86, even to 86∆HrpXG (mutant PXO86 with a damaged type III secretion system). These results indicated that SA plays an important role in disease resistance in rice plants, which can be clouded by high levels of endogenous SA and the use of particular rice varieties.

The bacterium Xanthomonas oryzae pv. Otyzae (Xoo) causes blight in rice worldwide, resulting in significant crop loss. However, no gene underlying a quantitative trait locus (QTL) for resistance against Xoo has been cloned yet. Here, we report the map-based cloning of a QTL, in which the NBS8R gene confers quantitative resistance to Xoo. NBS8R encodes an NB-ARC protein, which is involved in pathogen/microbe-associated molecular pattern-triggered immunity and whose expression is regulated by non-TAL effector XopQ-inducible Osa-miR1876 through DNA methylation. Sequence analysis of NBS8R in wild rice species and rice cultivars suggests that the Osa-miR1876 binding sites in the 5' UTR of NBS8R are inserted by chance and have undergone variations with Osa-miR1876 throughout evolution. The interaction between NBS8R and XopQ-inducible Osa-miR1876 is partially in keeping with the zigzag model, revealing that quantitative genes may also follow this model to control the innate immune response or basal disease resistance, and may prove valuable in utilizing the existing landraces that harbor the NBS8R gene but with no Osa-miR1876 binding site in rice breeding for bacterial blight resistance.

Development of marker-free and transgene insertion site-defined (MFTID) transgenic plants is essential for safe application of transgenic crops. However, MFTID plants have not been reported for wheat (Triticum aestivum). Here, we prepared a RNAi cassette for suppressing lipoxygenase (LOX) gene expression in wheat grains using a double right border T-DNA vector. The resultant construct was introduced into wheat genome via Agrobacterium-mediated transformation, with four homozygous marker-free transgenic lines (namely GLRW-1, -3, -5 and -8) developed. Aided by the newly published wheat genome sequence, the T-DNA insertion sites in GLRW-3 and GLRW-8 were elucidated at base-pair resolution. While the T-DNA in GLRW-3 inserted in an intergenic region, that of GLRW-8 inactivated an endogenous gene, which was thus excluded from further analysis. Compared to wild -type (WT) control, GLRW-1, -3 and -5 showed decreased LOX gene expression, lower LOX activity and less lipid peroxidation in the grains; they also exhibited significantly higher germination rates and better seedling growth after artificial ageing treatment. Interestingly, the three GLRW lines also had substantially increased contents of several fatty acids (e.g., linoleic acid and linolenic acid) in their grain and flour samples than WT control. Collectively, our data suggest that suppression of grain LOX activity can be employed to improve the storability and fatty acid content of wheat seeds and that the MFTID line GLRW-3 is likely of commercial value. Our approach may also be useful for developing the MFTID transgenic lines of other crops with enhanced grain storability and fatty acid content.

分子标记辅助选择育种己成为水稻分子育种的主要策略.明确目标基因在染色体上的物理图谱定位与功能标记的开发应用是开展分子标记辅助选择育种的前提条件.本研究对已报道的40个抗白叶枯病基因在染色体上的定位、分子标记详情进行了整理总结,并将31个水稻白叶枯病抗病基因在水稻基因组物理图谱上进行定位标注;此外,本研究还归纳xa5、xa13、Xa21、Xa27四个抗病基因的功能标记,新开发了抗性基因Xa10、Xa23的功能标记.本研究将给育种工作者更好地利用水稻抗白叶枯病基因与鉴定抗性材料提供便利.

BACKGROUND:Host genetic backgrounds affect gene functions. The genetic backgrounds of genetically engineered organisms must be identified to confirm their genetic backgrounds identity with those of recipients. Marker-assisted backcrossing (MAB), transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) editing are three commonly used genetic engineering techniques. However, methods for genetic background screening between genetically engineered organisms and corresponding recipients suffer from low efficiency, low accuracy or high cost.RESULTS:Here, we improved our previously reported AmpSeq-SSR method, an amplicon sequencing-based simple sequence repeat (SSR) genotyping method, by selecting SSR loci with high polymorphism among varieties. Ultimately, a set of 396 SSRs was generated and applied to evaluate the genetic backgrounds identity between rice lines developed through MAB, transgenesis, and CRISPR/Cas9 editing and the respective recipient rice. We discovered that the percentage of different SSRs between the MAB-developed rice line and its recipient was as high as 23.5%. In contrast, only 0.8% of SSRs were different between the CRISPR/Cas9-system-mediated rice line and its recipient, while no SSRs showed different genotypes between the transgenic rice line and its recipient. Furthermore, most differential SSRs induced by MAB technology were located in non-coding regions (62.9%), followed by untranslated regions (21.0%) and coding regions (16.1%). Trinucleotide repeats were the most prevalent type of altered SSR. Most importantly, all altered SSRs located in coding regions were trinucleotide repeats.CONCLUSIONS:This method is not only useful for the background evaluation of genetic resources but also expands our understanding of the unintended effects of different genetic engineering techniques. While the work we present focused on rice, this method can be readily extended to other organisms.

Table S2 Primers for the detection of 396 SSR loci. (XLSX 35 kb)

The pyramiding of resistance (R) genes provides broad-spectrum and durable resistance to plant diseases. However, the genetic basis for bacterial blight (BB) resistance remains unclear. The BB R gene pyramided line IRBB54, which expresses xa5 and Xa21, possessed a higher level of resistance than both single R gene lines. Large-scale genotyping of genetic markers in this study revealed similar genetic backgrounds among the near-isogenic lines (NILs), suggesting that resistance in the resistant NILs was mainly conferred by the individual R genes or the interaction between them. Transcriptome analysis demonstrated that more than 50% of the differentially expressed genes (DEGs), and more than 70% of the differentially expressed functions, were shared between IRBB54 and IRBB5 or IRBB21. Most of the DEGs in the resistant NILs were downregulated and are predicted to function in cellular and biological process. The DEGs common among the resistant NILs mainly showed non-additive expression patterns and enrichment in stress-related pathways. The differential expression of agronomic trait-controlled genes in the resistant NILs, especially in IRBB54, indicated the existence of potential side-effects resulting from gene pyramiding. Our findings contribute to the understanding of R gene pyramiding, as well as its effects on targeted and non-targeted trait(s).