Background Rice farming faces a significant challenge from the brown planthopper (BPH), a destructive pest that threatens crop yields. Developing BPH-resistant rice varieties is critical for ensuring food security. The pyramiding of BPH resistance genes, BPH14 and BPH15, has proven effective in providing protection in elite rice strains. MicroRNAs (miRNAs) play a pivotal role in plant defense by fine-tuning resistance responses through the modulation of genes involved in various signaling pathways and metabolic processes. B1415, the pyramiding line containing BPH14 and BPH15, exhibits stronger BPH resistance compared to its recurrent parent, Wushansimiao, without affecting other important agronomic traits. However, the molecular basis underlying the resistance conferred by the BPH14/BPH15 pyramiding rice remains largely unclear, particularly with respect to the potential regulatory role of miRNAs. Studying miRNAs in resistance gene pyramiding lines like B1415 is essential for advancing rice breeding efforts, as such research can uncover regulatory networks that enhance pest resistance and identify miRNA-mRNA interactions as potential targets for genetic manipulation. Results The study investigated miRNA levels in B1415 and their recurrent parent (RP) under BPH infestation employing high-throughput sequencing and revealed 136 differentially expressed miRNAs (DEMs) among 550 known miRNAs. An integrated analysis highlighted that 587 miRNA-mRNA pairs linking 95 DEMs to 537 targeted genes were enriched in phenylpropanoid and lignin metabolism, circadian rhythms, and amino acid metabolism. The candidate DEMs, miR172d-3p, and miR396 family members were identified as negative regulators to decrease their target genes Os06g0708700 (encoding a nodulin-like protein) and Os11g0129700 (encoding an AP2 domain transcription factor), suggesting their key roles in rice against BPH. Conclusions Our investigation provides the first insights into miRNA-mediated defense mechanisms in the B1415. Identifying miRNAs and their target mRNAs in BPH resistance opens a new avenue for rice breeding programs, offering potential targets for improving pest resistance. Understanding these molecular interactions paves the way for developing more resistant rice cultivars, thereby contributing to sustainable rice production and food security.

Proper anther dehiscence is essential for successful pollination and male fertility in flowering plants. However, many gaps remain in the regulatory mechanisms underlying anther dehiscence in plants, including a possible role for abscisic acid (ABA) in anther dehiscence. Here, we reported a member of 9-cis-epoxycarotenoid dioxygenase family (NCED), OsNCED5, which regulates proper anther dehiscence for male fertility in rice. OsNCED5 encodes a chloroplast-localized ABA biosynthetic enzyme highly expressed in anther during the late phase. Disruption of OsNCED5 by CRISPR/Cas9-mediated mutagenesis led to the reduction of ABA accumulation in anthers, thus the failure of secondary cell wall (SCW) thickening in anther endothecium due to loss of lignin biosynthesis, which eventually resulting in indehiscent anthers and significantly reduced fertility. Further analysis indicated that OsNCED5 probably confer a conserved fertility regulation mechanism between japonica and indica subspecies. Taken together, we have identified a key OsNCED regulator involved anther dehiscence -based male fertility, and provide a valuable genetic resource for fertility improvement in rice.

The AP2/ERF transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4) plays diverse roles in plant development and responses to abiotic stress. However, its potential involvement in regulating anthocyanin biosynthesis is not fully understood. In this study, three different loss-of-function abi4 alleles (abi4-1, abi4-2, and abi4-101) were employed to investigate the role of ABI4 in the regulation of anthocyanin accumulation in Arabidopsis seedlings. These abi4 mutants exhibited significantly increased anthocyanin accumulation, which was associated with elevated expression of genes involved in anthocyanin biosynthesis. HY5 (LONG HYPOCOTYL 5), a central component of photomorphogenesis, acts as a key light-regulated molecular switch. Further analysis revealed that ABI4 requires HY5 to function as a negative regulator of anthocyanin biosynthesis. Additionally, loss of ABI4 resulted in heightened light sensitivity, leading to increased light-induced chlorophyll accumulation and chloroplast development, along with upregulation of photosynthesis-related genes. Interestingly, the light-hypersensitive phenotype of abi4 mutants was partially rescued by the loss of HY5 function. Taken together, these findings demonstrate that ABI4 negatively regulates anthocyanin accumulation in Arabidopsis seedlings through a HY5-dependent light signaling pathway.

Autophagy is a highly conserved intracellular degradation system that is crucial for nutrient recycling, thus regulating plant growth and development as well as in response to various stresses. Halophytic plant Lycium ruthenicum Murray (L. ruthenicum) is considered as a potential model plant for studying the physiological mechanisms of salt stress tolerance in plants. Although the genome sequence of L. ruthenicum is available, the characteristics and functions of the salt stress-related genes remain largely unknown. In the present study, a total of 36 AuTophaGy-related (ATG) genes were identified in L. ruthenicum and detailed characteristics of them were given. Quantitative real-time polymerase chain reaction analysis revealed that the expression of 25 LrATGs was significantly upregulated after salt stress treatments. Furthermore, the autophagic marker line pSuper:GFP-LrATG8g was generated and used to demonstrate the salt stress-induced autophagy, as revealed by measuring autophagic flux and observing autophagosome formation. The pSuper:LrATG5-GFP overexpression (OE) lines were also generated and further phenotypic analysis showed that OE-LrATG8g and OE-LrATG5 plants exhibited better salt tolerance than that of WT plants. To the best of our knowledge, this study firstly reports a detailed overview of LrATGs-mediated autophagy in L. ruthenicum response to salt stress. These findings contribute to a global understanding of the characteristics of ATG genes in L. ruthenicum and lay a foundation for future functional study.

The anther culture-based breeding of rice is a plant tissue culture technique that utilizes rice pollen to rapidly obtain haploid plants. In comparison with traditional breeding methods, this technique shortens the breeding cycle and enables the quick generation of homozygous plants, which is of great significance for the development of new rice varieties and the expansion of germplasm resources. With the advancement of technologies, the use of the anther culture technique in rice breeding has matured and has been applied to the development and utilization of new varieties with high yield, multiple resistances, and superior quality, in combination with other breeding methods. This technique has gained widespread attention globally, with many countries adopting it to create new germplasm resources. This study reviews advances in the rice anther culture technique, the factors influencing anther culture efficiency, and the progress in breeding rice varieties using this technique, as well as analyzes the current challenges and future prospects of anther culture breeding.

Rice, a vital crop, faces significant threats from the brown planthopper (BPH), which impacts plant growth and yield. Pyramiding the BPH resistance genes BPH14 and BPH15 provides rice crops with reliable and lasting protection against BPH. Nonetheless, current research lacks clarity on the molecular processes responsible for BPH14/BPH15-mediated resistance to BPH. In this study, utilizing high-throughput metabolomics and integrating transcriptomic data, we investigated the metabolic adaptations of the BPH14/BPH15 pyramiding line (B1415) and its recurrent parent (RP) during early and late infestation stages. The analysis identified 1007 metabolites, mainly consisting of lipids and lipid-like molecules, together with phenylpropanoid and polyketide classes. Differentially accumulated metabolites (DAMs) displayed different patterns in B1415 and RP, particularly in flavonoid and phenylpropanoid biosynthesis pathways, which were more pronounced in the resistant B1415. Furthermore, ferulic acid (FA) was found to negatively regulate BPH resistance. These findings elucidate critical metabolic pathways involved in rice defense mechanisms and underscore the potential of B1415’s enhanced metabolic responses in conferring durable resistance against BPH.

The brown planthopper (Nilaparvata lugens Stål, BPH) is a destructive pest of rice. Non-coding RNAs (ncRNAs) regulate the defense mechanisms in rice and the adaptive strategies of BPHs. In rice, ncRNAs modulate key resistance pathways such as jasmonic acid biosynthesis, flavonoid production, and phenylpropanoid metabolism, which increases BPH resistance. In BPHs, ncRNAs regulate processes such as reproduction, metabolism, and wing polyphenism, which facilitate adaptation and virulence. Cross-kingdom interactions between rice and BPHs reveal the dynamic molecular interplay that underpins this pest–host relationship. These new insights into ncRNA functions will help improve innovative pest management strategies and equip rice varieties with enhanced BPH resistance.

Grain chalkiness is an undesirable agronomic trait that negatively affects both the yield and quality of rice (Oryza sativa). The molecular mechanisms underlying chalky grain phenotype have remained largely unclear. In this study, we selected the rice variety HK300 with a high chalkiness, and ZR24D with a low chalkiness, as experimental materials and systematically characterized the reasons of grain chalkiness formation at molecular level by means of RNA-seq analysis. Analysis results revealed that the differentially expressed genes (DEGs) in these two rice varieties were significantly enriched in transcriptional regulation, sucrose and starch metabolism, and phytohormone signal transduction. Moreover, we found the expression of 13 genes related to trehalose pathway (4 out of 14 TPS genes and 9 out of 13 TPP genes in rice genome) were significantly different between the two varieties, indicating trehalose synthesis pathways may contribute to the increased chalkiness formation. Notably, the number of DEGs associated with the signal transduction pathway for indole-3-acetic acid (IAA), which has been rarely studied for its involvement in chalkiness formation, was the highest among those associated with plant hormone signal transduction. Among them, the expression of two IAA receptor genes, OsAFB3 and OsAFB5, were significantly lower in HK300 than that in ZR24D through RNA-seq and qRT-PCR. Furthermore, we newly validated the two genes negatively regulated the formation of chalkiness through gene knockout. Our findings provided the theoretical basis and novel gene resources for molecular breeding aimed at improving rice quality.

RH (Rathu Heenati), an indica rice cultivar from Sri Lanka, is highly resistant to several different biotypes of brown planthopper (Nilaparvata lugens Stål, BPH). In contrast, another indica rice cultivar, TN1 (Taichung Native 1), is highly susceptible to all biotypes of BPH. To reveal the molecular biological mechanism of biotic and abiotic stress mediated by BPH suction in two rice cultivars, RH and TN1, the genome-wide gene expressional profiling of these two rice varieties at 6 h after BPH infestation and needle puncturing was conducted by performing transcriptome analysis. In total, six samples (T6C, T6N, T6B, R6C, R6N, and R6B) were collected after performing different treatments for this expressional analysis. The results provide important information for the further isolation of BPH-resistance genes from RH as well as research on the mechanism of BPH resistance in RH. The transcriptome results revealed that TN1 (a BPH-susceptible rice variety) and RH (a BPH-resistant rice variety) harbor different pathways that respond to the physical injury of acupuncture and BPH infestation. The susceptible variety TN1 is more sensitive to the physical damage caused by acupuncture, whereas in the resistant variety RH, the response to insect damage is more rapid and direct. By comparing the gene changes of various plant hormones between the two varieties under different stresses, it was found that, in addition to the traditional SA and ET pathways related to the feeding induction of piercing-sucking pests, the expression patterns of JAZ-related genes in the JA signaling pathway also changed under two stresses (acupuncture induction and brown planthopper feeding induction), while the IAA hormone pathway-related genes in the susceptible variety TN1 also changed significantly after brown planthopper feeding. Furthermore, the results of this study help us to understand the biological process of rice responses to BPH and provide a promising direction to identify BPH-resistance-related genes in RH.

As an important global food crop, rice is damaged by a variety of piercing–sucking pests. Identifying a broad-spectrum promoter induced by the physical signal of sucking pests and applying it to transgenic breeding to mitigate the damage caused by different sucking pests will significantly improve the efficiency of our breeding. This study compared the transcriptome changes in two rice varieties under needle-wounding stress to investigate their differential responses to mechanical damage. The results showed that the insect-susceptible variety TN1 exhibited more differentially expressed genes (DEGs) and greater changes in expression levels after needle treatment, indicating a more active internal gene regulatory network. GO and KEGG enrichment analysis further revealed that TN1 not only exhibited changes in genes related to the extracellular environment, but also mobilized more genes associated with stress response and defense. By screening the differentially expressed genes, we identified two promoters (P1 and P2) with inducible expression characteristics in both the resistant and susceptible rice varieties. These promoters were able to effectively drive the expression of the insect resistance gene OsLecRK1* and enhance the resistance of transgenic plants against the brown planthopper. This study provides promoter resources for the development of insect-resistant transgenic crops.

Powdery mildew fungi are obligate biotrophic pathogens that only invade plant epidermal cells. There are two epidermal surfaces in every plant leaf: the adaxial (upper) side and the abaxial (lower) side. While both leaf surfaces can be susceptible to adapted powdery mildew fungi in many plant species, there have been observations of leaf abaxial immunity in some plant species including Arabidopsis. The genetic basis of such leaf abaxial immunity remains unknown. In this study, we tested a series of Arabidopsis mutants defective in one or more known defense pathways with the adapted powdery mildew isolate Golovinomyces cichoracearum UCSC1. We found that leaf abaxial immunity was significantly compromised in mutants impaired for both the EDS1/PAD4- and PEN2/PEN3-dependent defenses. Consistently, expression of EDS1-yellow fluorescent protein and PEN2-green fluorescent protein fusions from their respective native promoters in the respective eds1-2 and pen2-1 mutant backgrounds was higher in the abaxial epidermal cells than in the adaxial epidermal cells. Altogether, our results indicate that leaf abaxial immunity against powdery mildew in Arabidopsis is at least partially due to enhanced EDS1/PAD4- and PEN2/PEN3-dependent defenses. Such transcriptionally pre-programmed defense mechanisms may underlie leaf abaxial immunity in other plant species such as hemp and may be exploited for engineering adaxial immunity against powdery mildew fungi in crop plants. The leaf adaxial side of Arabidopsis Col-0 is susceptible to an adapted powdery mildew, whereas the abaxial side is highly resistant thanks to the enhanced PEN2/PEN3- and EDS1/PAD4-dependent defense mechanisms.

The brown planthopper (BPH; Nilaparvata lugens Stål) is the most significant insect pest compromising rice production globally. Phytohormones, which are small organic compounds produced by plants, play a crucial role in regulating plant growth and development. Nevertheless, extensive research has established that phytohormones are essential in modulating plant defense against BPH. Plants can achieve equilibrium between growth and defense by utilizing the intricate network of phytohormone signaling pathways to initiate optimal and efficient defensive responses to insects. In this review, we primarily address the roles of phytohormones in conferring resistance against BPH, with a focus on hormone cross-talk. We also discuss the potential value of integrating hormones with other agricultural practices to enhance plant defense and agricultural yield, which highlights the significance of novel approaches for environment-friendly insect pest management.

Cold stress has a significantly negative effect on the growth, development, and yield of rice. However, the genetic basis for the differences in the cold tolerance of Xian/indica and Geng/japonica rice seedlings is still largely unknown. In this study, an RIL population was generated by crossing of the cold-tolerant japonica variety Nipponbare and the cold-sensitive indica variety WD16343 for BSA-seq analysis, and a major cold tolerance QTL qCTS11 was identified on chromosome 11. This locus was narrowed to the 584 kb region through fine mapping. Sequence alignment and expression analysis identified the cloned gene COLD11 and a novel cold-related gene OsCTS11. According to the reported functional variation of COLD11, Nipponbare (TCG + 3GCG)×2 presented more GCG repeats in the 1st exon than WD16343 (TCG + 3GCG), partially explaining the difference in cold tolerance between the parents. OsCTS11, encoding a stress enhanced protein based on phylogenetic analysis, was strongly induced by cold stress and located in the chloroplast and the nucleus. oscts11-mutant lines generated via CRISPR/Cas9 system improved the cold tolerance of rice seedlings. Our study not only reveals novel genetic loci associated with cold tolerance, but also provides potentially valuable gene resources for the cultivation of cold-tolerant rice.

Rice (Oryza sativa L.) is the staple food of more than half of Earth’s population. Brown planthopper (Nilaparvata lugens Stål, BPH) is a host-specific pest of rice responsible for inducing major losses in rice production. Utilizing host resistance to control N. lugens is considered to be the most cost-effective method. Therefore, the exploration of resistance genes and resistance mechanisms has become the focus of breeders’ attention. During the long-term co-evolution process, rice has evolved multiple mechanisms to defend against BPH infection, and BPHs have evolved various mechanisms to overcome the defenses of rice plants. More than 49 BPH-resistance genes/QTLs have been reported to date, and the responses of rice to BPH feeding activity involve various processes, including MAPK activation, plant hormone production, Ca2+ flux, etc. Several secretory proteins of BPHs have been identified and are involved in activating or suppressing a series of defense responses in rice. Here, we review some recent advances in our understanding of rice–BPH interactions. We also discuss research progress in controlling methods of brown planthoppers, including cultural management, trap cropping, and biological control. These studies contribute to the establishment of green integrated management systems for brown planthoppers.

Bacillus thuringiensis (Bt)-secreted crystal (Cry) toxins form oligomeric pores in host cell membranes and are a common element in generating insect-resistant transgenic crops. Although Cry toxin function has been well documented, cellular defences against pore-formation have not been as well developed. Elucidation of the processes underlying this defence, however, could contribute to the development of enhanced Bt crops. Here, we demonstrate that Cry1Ca-mediated downregulation of microRNA-7322-5p (miR-7322-5p), which binds to the 3 & PRIME; untranslated region of p38, negatively regulates the susceptibility of Chilo suppressalis to Cry1Ca. Moreover, Cry1Ca exposure enhanced phosphorylation of Hsp19, and hsp19 downregulation increased susceptibility to Cry1Ca. Further, Hsp19 phosphorylation occurs downstream of p38, and pull-down assays confirmed the interactions between Hsp19 and Cry1Ca, suggesting that activation of Hsp19 by the miR-7322-5p/p38/Hsp19 pathway promotes Cry1Ca sequestration. To assess the efficacy of targeting this pathway in planta, double-stranded RNA (dsRNA) targeting C. suppressalis p38 (dsp38) was introduced into a previously generated cry1Ca-expressing rice line (1CH1-2) to yield a single-copy cry1Ca/dsp38 rice line (p38-rice). Feeding on this rice line triggered a significant reduction in C. suppressalis p38 expression and the line was more resistant to C. suppressalis than 1CH1-2 in both short term (7-day) and continuous feeding bioassays as well as field trials. These findings provide new insights into invertebrate epithelium cellular defences and demonstrate a potential new pyramiding strategy for Bt crops.

Brown planthopper ( Nilaparvata lugens Stål, BPH) is one of the most destructive pests of rice. Non-coding RNA plays an important regulatory role in various biological processes. However, comprehensive identification and characterization of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in BPH-infested rice have not been performed. Here, we performed a genome-wide analysis of lncRNAs and circRNAs in BPH6 -transgenic (resistant, BPH6G) and Nipponbare (susceptible, NIP) rice plants before and after BPH feeding (early and late stage) via deep RNA-sequencing. A total of 310 lncRNAs and 129 circRNAs were found to be differentially expressed. To reveal the different responses of resistant and susceptible rice to BPH herbivory, the potential functions of these lncRNAs and circRNAs as competitive endogenous RNAs (ceRNAs) were predicted and investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Dual-luciferase reporter assays revealed that miR1846c and miR530 were targeted by the lncRNAs XLOC_042442 and XLOC_028297, respectively. In responsive to BPH infestation, 39 lncRNAs and 21 circRNAs were predicted to combine with 133 common miRNAs and compete for miRNA binding sites with 834 mRNAs. These mRNAs predictably participated in cell wall organization or biogenesis, developmental growth, single-organism cellular process, and the response to stress. This study comprehensively identified and characterized lncRNAs and circRNAs, and integrated their potential ceRNA functions, to reveal the rice BPH-resistance network. These results lay a foundation for further study on the functions of lncRNAs and circRNAs in the rice-BPH interaction, and enriched our understanding of the BPH-resistance response in rice.

Introduction:The brown planthopper (Nilaparvata lugens Stål, BPH) is one of the most economically significant pests of rice. The Bph30 gene has been successfully cloned and conferred rice with broad-spectrum resistance to BPH. However, the molecular mechanisms by which Bph30 enhances resistance to BPH remain poorly understood.Methods:Here, we conducted a transcriptomic and metabolomic analysis of Bph30-transgenic (BPH30T) and BPH-susceptible Nipponbare plants to elucidate the response of Bph30 to BPH infestation.Results:Transcriptomic analyses revealed that the pathway of plant hormone signal transduction enriched exclusively in Nipponbare, and the greatest number of differentially expressed genes (DEGs) were involved in indole 3-acetic acid (IAA) signal transduction. Analysis of differentially accumulated metabolites (DAMs) revealed that DAMs involved in the amino acids and derivatives category were down-regulated in BPH30T plants following BPH feeding, and the great majority of DAMs in flavonoids category displayed the trend of increasing in BPH30T plants; the opposite pattern was observed in Nipponbare plants. Combined transcriptomics and metabolomics analysis revealed that the pathways of amino acids biosynthesis, plant hormone signal transduction, phenylpropanoid biosynthesis and flavonoid biosynthesis were enriched. The content of IAA significantly decreased in BPH30T plants following BPH feeding, and the content of IAA remained unchanged in Nipponbare. The exogenous application of IAA weakened the BPH resistance conferred by Bph30.Discussion:Our results indicated that Bph30 might coordinate the movement of primary and secondary metabolites and hormones in plants via the shikimate pathway to enhance the resistance of rice to BPH. Our results have important reference significance for the resistance mechanisms analysis and the efficient utilization of major BPH-resistance genes.

The brown planthopper (BPH) (Nilaparvata lugens) sucks rice sap causing leaves to turn yellow and wither, often leading to reduced or zero yields. Rice co-evolved to resist damage by BPH. However, the molecular mechanisms, including the cells and tissues, involved in the resistance are still rarely reported. Single-cell sequencing technology allows us to analyze different cell types involved in BPH resistance. Here, using single-cell sequencing technology, we compared the response offered by the leaf sheaths of the susceptible (TN1) and resistant (YHY15) rice varieties to BPH (48 hours after infestation). We found that the 14,699 and 16,237 cells (identified via transcriptomics) in TN1 and YHY15 could be annotated using cell-specific marker genes into nine cell-type clusters. The two rice varieties showed significant differences in cell types (such as mestome sheath cells, guard cells, mesophyll cells, xylem cells, bulliform cells, and phloem cells) in the rice resistance mechanism to BPH. Further analysis revealed that although mesophyll, xylem, and phloem cells are involved in the BPH resistance response, the molecular mechanism used by each cell type is different. Mesophyll cell may regulate the expression of genes related to vanillin, capsaicin, and ROS production, phloem cell may regulate the cell wall extension related genes, and xylem cell may be involved in BPH resistance response by controlling the expression of chitin and pectin related genes. Thus, rice resistance to BPH is a complicated process involving multiple insect resistance factors. The results presented here will significantly promote the investigation of the molecular mechanisms underlying the resistance of rice to insects and accelerate the breeding of insect-resistant rice varieties.