OsEIL2 balances rice immune responses against (hemi)biotrophic and necrotrophic pathogens via the salicylic acid and jasmonic acid synergism.

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.

Antagonistic control of rice immunity against distinct pathogens by the two transcription modules via salicylic acid and jasmonic acid pathways.

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.

Single-cell transcriptome analysis dissects lncRNA-associated gene networks in Arabidopsis.

The plant genome produces an extremely large collection of long noncoding RNAs (lncRNAs) that are generally expressed in a context-specific manner and have pivotal roles in regulation of diverse biological processes. Here, we mapped the transcriptional heterogeneity of lncRNAs and their associated gene regulatory networks at single-cell resolution. We generated a comprehensive cell atlas at the whole-organism level by integrative analysis of 28 published single-cell RNA sequencing (scRNA-seq) datasets from juvenile Arabidopsis seedlings. We then provided an in-depth analysis of cell-type-related lncRNA signatures that show expression patterns consistent with canonical protein-coding gene markers. We further demonstrated that the cell-type-specific expression of lncRNAs largely explains their tissue specificity. In addition, we predicted gene regulatory networks on the basis of motif enrichment and co-expression analysis of lncRNAs and mRNAs, and we identified putative transcription factors orchestrating cell-type-specific expression of lncRNAs. The analysis results are available at the single-cell-based plant lncRNA atlas database (scPLAD; https://biobigdata.nju.edu.cn/scPLAD/). Overall, this work demonstrates the power of integrative single-cell data analysis applied to plant lncRNA biology and provides fundamental insights into lncRNA expression specificity and associated gene regulation.

Osmotin1 is involved in rice resistance to Schizotetranychus oryzae (Acari: Tetranychidae) infestation.

BACKGROUND: Rice is one of the most consumed cereals in the world. Productivity losses are caused by different biotic stresses. One of the most common is the phytophagous mite Schizotetranychus oryzae Rossi de Simons (Acari: Tetranychidae), which inhibits plant development and seed production. The identification of plant defense proteins is important for a better understanding of the mite-plant interaction. We previously detected a high expression of Osmotin1 protein in mite-resistant rice cultivars, under infested conditions, suggesting it could be involved in plant defense against mite attack. We therefore aimed to evaluate the responses of three rice lines overexpressing Osmotin1 (OSM1-OE) and three lines lacking the Osmotin1 gene (osm1-ko) to mite attack. RESULTS: The numbers of individuals (adults, immature stages, and eggs) were significantly lower in OSM1-OE lines than those in wild-type (WT) plants. On the other hand, the osm1-ko lines showed larger numbers of mites per leaf than WT plants. When plants reached the full maturity stage, two out of the three infested OSM1-OE lines presented lower plant height than WT, while the three osm1-ko lines (infested or not) presented higher plant height than WT. The reduction in seed number caused by mite infestation was lower in OSM1-OE lines (12-19%) than in WT plants (34%), while osm1-ko lines presented higher reduction (24-54%) in seed number than WT plants (13%). CONCLUSION: These data suggest that Osmotin1 is involved in rice resistance to S. oryzae infestation. This is the first work showing increased plant resistance to herbivory overexpressing an Osmotin gene. © 2023 Society of Chemical Industry.

Fine mapping and candidate gene analysis of qSB12YSB, a gene conferring major quantitative resistance to rice sheath blight.

KEY MESSAGE: qSB12YSB, a major quantitative sheath blight resistance gene originated from rice variety YSBR1 with good breeding potential, was mapped to a 289-Kb region on chromosome 12. Sheath blight (ShB), caused by Rhizoctonia solani kühn, is one of the most serious global rice diseases. Rice resistance to ShB is a typical of quantitative trait controlled by multiple quantitative trait loci (QTLs). Many QTLs for ShB resistance have been reported while only few of them were fine-mapped. In this study, we identified a QTL on chromosome 12, in which the qSB12YSB resistant allele shows significant ShB resistance, by using 150 BC4 backcross inbred lines employing the resistant rice variety YSBR1 as the donor and the susceptible variety Lemont (LE) as the recurrent parent. We further fine-mapped qSB12YSB to a 289-kb region by generating 34 chromosomal segment substitution lines and identified a total of 18 annotated genes as the most likely candidates for qSB12YSB after analyzing resequencing and transcriptomic data. KEGG analysis suggested that qSB12YSB might activate secondary metabolites biosynthesis and ROS scavenging system to improve ShB resistance. qSB12YSB conferred significantly stable resistance in three commercial rice cultivars (NJ9108, NJ5055 and NJ44) in field trials when introduced through marker assisted selection. Under severe ShB disease conditions, qSB12YSB significantly reduced yield losses by up to 13.5% in the LE background, indicating its great breeding potential. Our results will accelerate the isolation of qSB12YSB and its utilization in rice breeding programs against ShB.

Knockout of transcription factor OsERF65 enhances ROS scavenging ability and confers resistance to rice sheath blight.

Rice sheath blight (ShB) is a devastating disease that severely threatens rice production worldwide. Induction of cell death represents a key step during infection by the ShB pathogen Rhizoctonia solani. Nonetheless, the underlying mechanisms remain largely unclear. In the present study, we identified a rice transcription factor, OsERF65, that negatively regulates resistance to ShB by suppressing cell death. OsERF65 was significantly upregulated by R. solani infection in susceptible cultivar Lemont and was highly expressed in the leaf sheath. Overexpression of OsERF65 (OsERF65OE) decreased rice resistance, while the knockout mutant (oserf65) exhibited significantly increased resistance against ShB. The transcriptome assay revealed that OsERF65 repressed the expression of peroxidase genes after R. solani infection. The antioxidative enzyme activity was significantly increased in oserf65 plants but reduced in OsERF65OE plants. Consistently, hydrogen peroxide content was apparently reduced in oserf65 plants but accumulated in OsERF65OE plants. OsERF65 directly bound to the GCC box in the promoter regions of four peroxidase genes and suppressed their transcription, reducing the ability to scavenge reactive oxygen species (ROS). The oserf65 mutant exhibited a slight decrease in plant height but increased grain yield. Overall, our results revealed an undocumented role of OsERF65 that acts as a crucial regulator of rice resistance to R. solani and a potential target for improving both ShB resistance and rice yield.

Genome-Wide Association Study Identifies a Plant-Height-Associated Gene OsPG3 in a Population of Commercial Rice Varieties.

Plant height is one of the most crucial components of plant structure. However, due to its complexity, the genetic architecture of rice plant height has not been fully elucidated. In this study, we performed a genome-wide association study (GWAS) to determine rice plant height using 178 commercial rice varieties and identified 37 loci associated with rice plant height (LAPH). Among these loci, in LAPH2, we identified a polygalacturonase gene, OsPG3, which was genetically and functionally associated with rice plant height. The rice plant exhibits a super dwarf phenotype when the knockout of the OsPG3 gene occurs via CRISPR-Cas9 gene-editing technology. RNA-Seq analysis indicated that OsPG3 modulates the expression of genes involved in phytohormone metabolism and cell-wall-biosynthesis pathways. Our findings suggest that OsPG3 plays a vital role in controlling rice plant height by regulating cell wall biosynthesis. Given that rice architecture is one of the most critical phenotypes in rice breeding, OsPG3 has potential in rice's molecular design breeding toward an ideal plant height.

Identification of two novel rice S genes through combination of association and transcription analyses with gene-editing technology.

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.

Approaches to Reduce Rice Blast Disease Using Knowledge from Host Resistance and Pathogen Pathogenicity.

Rice is one of the staple foods for the majority of the global population that depends directly or indirectly on it. The yield of this important crop is constantly challenged by various biotic stresses. Rice blast, caused by Magnaporthe oryzae (M. oryzae), is a devastating rice disease causing severe yield losses annually and threatening rice production globally. The development of a resistant variety is one of the most effective and economical approaches to control rice blast. Researchers in the past few decades have witnessed the characterization of several qualitative resistance (R) and quantitative resistance (qR) genes to blast disease as well as several avirulence (Avr) genes from the pathogen. These provide great help for either breeders to develop a resistant variety or pathologists to monitor the dynamics of pathogenic isolates, and ultimately to control the disease. Here, we summarize the current status of the isolation of R, qR and Avr genes in the rice-M. oryzae interaction system, and review the progresses and problems of these genes utilized in practice for reducing rice blast disease. Research perspectives towards better managing blast disease by developing a broad-spectrum and durable blast resistance variety and new fungicides are also discussed.

Suppression of Rice Osa-miR444.2 Improves the Resistance to Sheath Blight in Rice Mediating through the Phytohormone Pathway.

MicroRNAs (miRNAs) are a class of conserved small RNA with a length of 21-24 nucleotides in eukaryotes, which are involved in development and defense responses against biotic and abiotic stresses. By RNA-seq, Osa-miR444b.2 was identified to be induced after Rhizoctonia solani (R. solani) infection. In order to clarify the function of Osa-miR444b.2 responding to R. solani infection in rice, transgenic lines over-expressing and knocking out Osa-miR444b.2 were generated in the background of susceptible cultivar Xu3 and resistant cultivar YSBR1, respectively. Over-expressing Osa-miR444b.2 resulted in compromised resistance to R. solani. In contrast, the knocking out Osa-miR444b.2 lines exhibited improved resistance to R. solani. Furthermore, knocking out Osa-miR444b.2 resulted in increased height, tillers, smaller panicle, and decreased 1000-grain weight and primary branches. However, the transgenic lines over-expressing Osa-miR444b.2 showed decreased primary branches and tillers, but increased panicle length. These results indicated that Osa-miR444b.2 was also involved in regulating the agronomic traits in rice. The RNA-seq assay revealed that Osa-miR444b.2 mainly regulated the resistance to rice sheath blight disease by affecting the expression of plant hormone signaling pathways-related genes such as ET and IAA, and transcription factors such as WRKYs and F-boxes. Together, our results suggest that Osa-miR444b.2 negatively mediated the resistance to R. solani in rice, which will contribute to the cultivation of sheath blight resistant varieties.

Identification of Elite R-Gene Combinations against Blast Disease in Geng Rice Varieties.

Rice blast, caused by the Magnaporthe oryzae fungus, is one of the most devastating rice diseases worldwide. Developing resistant varieties by pyramiding different blast resistance (R) genes is an effective approach to control the disease. However, due to complex interactions among R genes and crop genetic backgrounds, different R-gene combinations may have varying effects on resistance. Here, we report the identification of two core R-gene combinations that will benefit the improvement of Geng (Japonica) rice blast resistance. We first evaluated 68 Geng rice cultivars at seedling stage by challenging with 58 M. oryzae isolates. To evaluate panicle blast resistance, we inoculated 190 Geng rice cultivars at boosting stage with five groups of mixed conidial suspensions (MCSs), with each containing 5-6 isolates. More than 60% cultivars displayed moderate or lower levels of susceptibility to panicle blast against the five MCSs. Most cultivars contained two to six R genes detected by the functional markers corresponding to 18 known R genes. Through multinomial logistics regression analysis, we found that Pi-zt, Pita, Pi3/5/I, and Pikh loci contributed significantly to seedling blast resistance, and Pita, Pi3/5/i, Pia, and Pit contributed significantly to panicle blast resistance. For gene combinations, Pita+Pi3/5/i and Pita+Pia yielded more stable pyramiding effects on panicle blast resistance against all five MCSs and were designated as core R-gene combinations. Up to 51.6% Geng cultivars in the Jiangsu area contained Pita, but less than 30% harbored either Pia or Pi3/5/i, leading to less cultivars containing Pita+Pia (15.8%) or Pita+Pi3/5/i (5.8%). Only a few varieties simultaneously contained Pia and Pi3/5/i, implying the opportunity to use hybrid breeding procedures to efficiently generate varieties with either Pita+Pia or Pita+Pi3/5/i. This study provides valuable information for breeders to develop Geng rice cultivars with high resistance to blast, especially panicle blast.

Ectopic Expression of Gastrodia Antifungal Protein in Rice Enhances Resistance to Rice Sheath Blight Disease.

Sheath blight (ShB) disease, caused by Rhizoctonia solani Kühn, is one of the most serious rice diseases. Rice breeding against ShB has been severely hindered because no major resistance genes or germplasms are available in rice. Here, we report that introduction of Gastrodia antifungal protein (GAFP) genes from Gastrodia elata B1 into rice significantly enhances resistance to rice ShB. Four GAFP genes were cloned from G. elata B1, and all displayed a strong ability to inhibit R. solani growth in plate assays. Two versions, with or without a signal peptide, for each of the four GAFP genes were introduced into XD3 and R6547 rice cultivars, and all transgenic lines displayed stronger ShB resistance than the corresponding wild-type control in both greenhouse and field conditions. Importantly, GAFP2 showed the highest ShB resistance; GAFPs with and without its signal peptide showed no significant differences in enhancing ShB resistance. We also evaluated the agronomic traits of these transgenic rice and found that ectopic expression of GAFPs in rice at appropriate levels did not affect agronomic traits other than enhancing ShB resistance. Together, these results indicate that GAFP genes, especially GAFP2, have great potential in rice breeding against ShB disease.

Nanosheet-Facilitated Spray Delivery of dsRNAs Represents a Potential Tool to Control Rhizoctonia solani Infection.

Rhizoctonia solani is one of the important pathogenic fungi causing several serious crop diseases, such as maize and rice sheath blight. Current methods used to control the disease mainly depend on spraying fungicides because there is no immunity or high resistance available in crops. Spraying double-strand RNA (dsRNA) for induced-gene silencing (SIGS) is a new potentially sustainable and environmentally friendly tool to control plant diseases. Here, we found that fluorescein-labelled EGFP-dsRNA could be absorbed by R. solani in co-incubation. Furthermore, three dsRNAs, each targeting one of pathogenicity-related genes, RsPG1, RsCATA, and RsCRZ1, significantly downregulated the transcript levels of the target genes after co-incubation, leading to a significant reduction in the pathogenicity of the fungus. Only the spray of RsCRZ1 dsRNA, but not RsPG1 or RsCATA dsRNA, affected fungal sclerotium formation. dsRNA stability on leaf surfaces and its efficiency in entering leaf cells were significantly improved when dsRNAs were loaded on layered double hydroxide (LDH) nanosheets. Notably, the RsCRZ1-dsRNA-LDH approach showed stronger and more lasting effects than using RsCRZ1-dsRNA alone in controlling pathogen development. Together, this study provides a new potential method to control crop diseases caused by R. solani.

Correlations among population genetic structure, geographic origin, growth rate, and fungicide resistance of Rhizoctonia solani AG 1-IA, the pathogen of rice sheath blight.

Rhizoctonia solani AG1-IA is a necrotrophic fungus that causes rice sheath blight and results in severe yield and quality reductions in rice worldwide. Differences of genetic structure and fungicide sensitivity of the pathogen have significant effects on the severity and control effect of this disease in the field. To determine correlations among population genetic structure, geographic origin, growth rate, and fungicide resistance of the pathogen, 293 strains of R. solani were isolated from diseased rice collected from 13 cities of Jiangsu Province and five regions of China. Simple sequence repeat (SSR) molecular marker technology was used to analyze the genetic diversity of these strains, and a total of 74 bands were amplified by nine pairs of primers. Population genetic structure analysis showed that strains from Central China and northern Jiangsu had the highest Nei's gene diversity index and Shannon diversity index. The vast majority of strains grew fast with colony diameters of more than 60.0 mm cultured at 28 °C for 36 h. The half-maximal effective concentration (EC50) of them to tebuconazole, thifluzamide, and propiconazole varied ∼16.2-, 3.8-, and 7.5-fold. However, the genetic diversity of R. solani had no significant correlation with their geographic origin, growth rate or fungicide sensitivity.

Development of Rice Variety With Durable and Broad-Spectrum Resistance to Blast Disease Through Marker-Assisted Introduction of Pigm Gene.

Rice blast, caused by Magnaporthe oryzae (M. oryzae), is one of the most destructive diseases threatening rice production worldwide. Development of resistant cultivars using broad-spectrum resistance (R) genes with high breeding value is the most effective and economical approach to control this disease. In this study, the breeding potential of Pigm gene in geng/japonica rice breeding practice in Jiangsu province was comprehensively evaluated. Through backcross and marker-assisted selection (MAS), Pigm was introduced into two geng rice cultivars (Wuyungeng 32/WYG32 and Huageng 8/HG8). In each genetic background, five advanced backcross lines with Pigm (ABLs) and the same genotypes as the respective recurrent parent in the other 13 known R gene loci were developed. Compared with the corresponding recurrent parent, all these ABLs exhibited stronger resistance in seedling inoculation assay using 184 isolates collected from rice growing regions of the lower region of the Yangtze River. With respect to panicle blast resistance, all ABLs reached a high resistance level to blast disease in tests conducted in three consecutive years with the inoculation of seven mixed conidial suspensions collected from different regions of Jiangsu province. In natural field nursery assays, the ABLs showed significantly higher resistance than the recurrent parents. No common change on importantly morphological traits and yield-associated components was found among the ABLs, demonstrating the introduction of Pigm had no tightly linked undesirable effect on rice economically important traits and its associated grain weight reduction effect could be probably offset by others grain weight genes or at least in the background of the aforementioned two varieties. Notably, one rice line with Pigm, designated as Yangnonggeng 3091, had been authorized as a new variety in Jiangsu province in 2021, showing excellent performance on both grain yield and quality, as well as the blast resistance. Together, these results suggest that the Pigm gene has a high breeding value in developing rice varieties with durable and broad-spectrum resistance to blast disease.

A VQ-motif-containing protein fine-tunes rice immunity and growth by a hierarchical regulatory mechanism.

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.