Rice microRNA156/529-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7/14/17 modules regulate defenses against bacteria.

Rice (Oryza sativa L.) microRNA156/529-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7/14/17 (miR156/529-SPL7/14/17) modules have pleiotropic effects on many biological pathways. OsSPL7/14 can interact with DELLA protein SLENDER RICE1 (SLR1) to modulate gibberellin acid (GA) signal transduction against the bacterial pathogen Xanthomonas oryzae pv. oryzae. However, whether the miR156/529-OsSPL7/14/17 modules also regulate resistance against other pathogens is unclear. Notably, OsSPL7/14/17 functioning as transcriptional activators, their target genes, and the corresponding downstream signaling pathways remain largely unexplored. Here, we demonstrate that miR156/529 play negative roles in plant immunity and that miR156/529-regulated OsSPL7/14/17 confer broad-spectrum resistance against 2 devastating bacterial pathogens. Three OsSPL7/14/17 proteins directly bind to the promoters of rice Allene Oxide Synthase 2 (OsAOS2) and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (OsNPR1) and activate their transcription, regulating jasmonic acid (JA) accumulation and the salicylic acid (SA) signaling pathway, respectively. Overexpression of OsAOS2 or OsNPR1 impairs the susceptibility of the osspl7/14/17 triple mutant. Exogenous application of JA enhances resistance of the osspl7/14/17 triple mutant and the miR156 overexpressing plants. In addition, genetic evidence confirms that bacterial pathogen-activated miR156/529 negatively regulate pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses, such as pattern recognition receptor Xa3/Xa26-initiated PTI. Our findings demonstrate that bacterial pathogens modulate miR156/529-OsSPL7/14/17 modules to suppress OsAOS2-catalyzed JA accumulation and the OsNPR1-promoted SA signaling pathway, facilitating pathogen infection. The uncovered miR156/529-OsSPL7/14/17-OsAOS2/OsNPR1 regulatory network provides a potential strategy to genetically improve rice disease resistance.

ENHANCED DISEASE SUSCEPTIBILITY 1 promotes hydrogen peroxide scavenging to enhance rice thermotolerance.

Heat stress is a major factor limiting the production and geographic distribution of rice (Oryza sativa), and breeding rice varieties with tolerance to heat stress is of immense importance. Although extensive studies have revealed that reactive oxygen species (ROS) play a critical role in rice acclimation to heat stress, the molecular basis of rice controlling ROS homeostasis remains largely unclear. In this study, we discovered a novel heat-stress-responsive strategy that orchestrates ROS homeostasis centering on an immune activator, rice ENHANCED DISEASE SUSCEPTIBILITY 1 (OsEDS1). OsEDS1, which confers heat stress tolerance, promotes hydrogen peroxide (H2O2) scavenging by stimulating catalase activity through the OsEDS1-catalase association. The loss-of-function mutation in OsEDS1 causes increased sensitivity to heat stress, whereas the overexpression of OsEDS1 enhances thermotolerance. Furthermore, overexpression lines greatly improved rice tolerance to heat stress during the reproductive stage, which was associated with substantially increased seed setting, grain weight, and plant yield. Rice CATALASE C (OsCATC), whose activity is promoted by OsEDS1, degrades H2O2 to activate rice heat stress tolerance. Our findings greatly expand our understanding of heat stress responses in rice. We reveal a molecular framework that promotes heat tolerance through ROS homeostasis regulation, suggesting a theoretical basis and providing genetic resources for breeding heat-tolerant rice varieties.

Regulatory network of rice in response to heat stress and its potential application in breeding strategy.

The rapid development of global industrialization has led to serious environmental problems, among which global warming has become one of the major concerns. The gradual rise in global temperature resulted in the loss of food production, and hence a serious threat to world food security. Rice is the main crop for approximately half of the world's population, and its geographic distribution, yield, and quality are frequently reduced due to elevated temperature stress, and breeding rice varieties with tolerance to heat stress is of immense significance. Therefore, it is critical to study the molecular mechanism of rice in response to heat stress. In the last decades, large amounts of studies have been conducted focusing on rice heat stress response. Valuable information has been obtained, which not only sheds light on the regulatory network underlying this physiological process but also provides some candidate genes for improved heat tolerance breeding in rice. In this review, we summarized the studies in this field. Hopefully, it will provide some new insights into the mechanisms of rice under high temperature stress and clues for future engineering breeding of improved heat tolerance rice.

The versatile functions of OsALDH2B1 provide a genic basis for growth-defense trade-offs in rice.

In plants, enhanced defense often compromises growth and development, which is regarded as trade-offs between growth and defense. Here we identified a gene, OsALDH2B1, that functions as a master regulator of the growth-defense trade-off in rice. OsALDH2B1 has its primary function as an aldehyde dehydrogenase and a moonlight function as a transcriptional regulator. Loss of function of OsALDH2B1 greatly enhanced resistance to broad-spectrum pathogens, including fungal blast, bacterial leaf blight, and leaf streak, but caused severe phenotypic changes such as male sterility and reduced plant size, grain size, and number. We showed that its primary function as a mitochondrial aldehyde dehydrogenase conditions male fertility. Its moonlight function of transcriptional regulation, featuring both repressing and activating activities, regulates a diverse range of biological processes involving brassinolide, G protein, jasmonic acid, and salicylic acid signaling pathways. Such regulations cause large impacts on the morphology and immunity of rice plants. The versatile functions of OsALDH2B1 provide an example of the genic basis of growth-defense trade-offs in plants.

Advances of Apetala2/Ethylene Response Factors in Regulating Development and Stress Response in Maize.

Apetala2/ethylene response factor (AP2/ERF) is one of the largest families of transcription factors, regulating growth, development, and stress response in plants. Several studies have been conducted to clarify their roles in Arabidopsis and rice. However, less research has been carried out on maize. In this review, we systematically identified the AP2/ERFs in the maize genome and summarized the research progress related to AP2/ERF genes. The potential roles were predicted from rice homologs based on phylogenetic and collinear analysis. The putative regulatory interactions mediated by maize AP2/ERFs were discovered according to integrated data sources, implying that they involved complex networks in biological activities. This will facilitate the functional assignment of AP2/ERFs and their applications in breeding strategy.

Knock out of transcription factor WRKY53 thickens sclerenchyma cell walls, confers bacterial blight resistance.

Plant cell walls are the first physical barrier against pathogen invasion, and plants thicken the cell wall to strengthen it and restrain pathogen infection. Bacterial blight is a devastating rice (Oryza sativa) disease caused by Xanthomonas oryzae pv. oryzae (Xoo), which typically enters the rice leaf through hydathodes and spreads throughout the plant via the xylem. Xoo interacts with cells surrounding the xylem vessel of a vascular bundle, but whether rice strengthens the sclerenchyma cell walls to stop pathogen proliferation is unclear. Here, we found that a WRKY protein, OsWRKY53, negatively confers resistance to Xoo by strengthening the sclerenchyma cell walls of the vascular bundle. OsMYB63 acts as a transcriptional activator and promotes the expression of three secondary cell wall-related cellulose synthase genes to boost cellulose accumulation, resulting in thickened sclerenchyma cell walls. Both OsWRKY53 and OsMYB63 are abundantly expressed in sclerenchyma cells of leaf vascular bundles. OsWRKY53 functions as a transcriptional repressor and acts genetically upstream of OsMYB63 to suppress its expression. The OsWRKY53-overexpressing and OsMYB63 knockout plants had thinner sclerenchyma cell walls, showing susceptibility to Xoo, while the OsWRKY53 knockout and OsMYB63-overexpressing plants had thicker sclerenchyma cell walls, exhibiting resistance to Xoo. These results suggest that modifying these candidate genes provides a strategy to improve rice resistance to bacterial pathogens.

Hd3a and OsFD1 negatively regulate rice resistance to Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola.

In rice, Hd3a, GF14 and OsFD1 proteins, forming florigen activation complex, are key components in flowering time regulation. GF14 genes in rice response to biotic and abiotic stress has also been well addressed. The role of GF14e in rice defense has been well studied. However, whether Hd3a and OsFD1 play roles in defense is unclear. In present study, we identified that Hd3a and OsFD1 expression is repressed by Xoo and JA, and validated that Hd3a and OsFD1 negatively regulate resistance to Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc). hd3a and osfd1 mutants increase resistance to Xoo and Xoc, and activate JA responsive genes expression. Our data also demonstrate that OsFD1 binds to the promoters of and activates the expression of JAZ genes; Hd3a, cooperating with GF14e, promotes OsFD1 action on JAZ gene expression. The functional confirmation of Hd3a and OsFD1 in rice defense makes them to be promising targets in molecular rice breeding.

Advances in understanding broad-spectrum resistance to pathogens in rice.

Rice diseases caused by multiple pathogen species are a major obstacle to achieving optimal yield. Using host pathogen species-non-specific broad-spectrum resistance (BSR) for rice improvement is an efficient way to control diseases. Recent advances in rice genomics and improved understanding of the mechanisms of rice-pathogen interactions have shown that using a single gene to improve rice BSR to multiple pathogen species is technically possible and the necessary resources exist. A variety of rice genes, including major disease resistance genes and defense-responsive genes, which function in pattern-triggered immunity signaling, effector-triggered immunity signaling or quantitative resistance, can mediate BSR to two or more pathogen species independently. These genes encode diverse proteins and function differently in promoting disease resistance, thus providing a relatively broad choice for different breeding programs. This updated knowledge will facilitate rice improvement with pathogen species-non-specific BSR via gene marker-assisted selection or biotechnological approaches.

Rice OsPAD4 functions differently from Arabidopsis AtPAD4 in host-pathogen interactions.

The extensively studied Arabidopsis phytoalexin deficient 4 (AtPAD4) gene plays an important role in Arabidopsis disease resistance; however, the function of its sequence ortholog in rice is unknown. Here, we show that rice OsPAD4 appears not to be the functional ortholog of AtPAD4 in host-pathogen interactions, and that the OsPAD4 encodes a plasma membrane protein but that AtPAD4 encodes a cytoplasmic and nuclear protein. Suppression of OsPAD4 by RNA interference (RNAi) increased rice susceptibility to the biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo), which causes bacteria blight disease in local tissue. OsPAD4-RNAi plants also show compromised wound-induced systemic resistance to Xoo. The increased susceptibility to Xoo was associated with reduced accumulation of jasmonic acid (JA) and phytoalexin momilactone A (MOA). Exogenous application of JA complemented the phenotype of OsPAD4-RNAi plants in response to Xoo. The following results suggest that OsPAD4 functions differently than AtPAD4 in response to pathogen infection. First, OsPAD4 plays an important role in wound-induced systemic resistance, whereas AtPAD4 mediates systemic acquired resistance. Second, OsPAD4-involved defense signaling against Xoo is JA-dependent, but AtPAD4-involved defense signaling against biotrophic pathogens is salicylic acid-dependent. Finally, OsPAD4 is required for the accumulation of terpenoid-type phytoalexin MOA in rice-bacterium interactions, but AtPAD4-mediated resistance is associated with the accumulation of indole-type phytoalexin camalexin.

Identifying RNA Modifications by Direct RNA Sequencing Reveals Complexity of Epitranscriptomic Dynamics in Rice.

Transcriptome analysis based on high-throughput sequencing of a cDNA library has been widely applied to functional genomic studies. However, the cDNA dependence of most RNA sequencing techniques constrains their ability to detect base modifications on RNA, which is an important element for the post-transcriptional regulation of gene expression. To comprehensively profile the N6-methyladenosine (m6A) and N5-methylcytosine (m5C) modifications on RNA, direct RNA sequencing (DRS) using the latest Oxford Nanopore Technology was applied to analyze the transcriptome of six tissues in rice. Approximately 94 million reads were generated, with an average length ranging from 619 nt to 1013 nt, and a total of 45,707 transcripts across 34,763 genes were detected. Expression profiles of transcripts at the isoform level were quantified among tissues. Transcriptome-wide mapping of m6A and m5C demonstrated that both modifications exhibited tissue-specific characteristics. The transcripts with m6A modifications tended to be modified by m5C, and the transcripts with modifications presented higher expression levels along with shorter poly(A) tails than transcripts without modifications, suggesting the complexity of gene expression regulation. Gene Ontology analysis demonstrated that m6A- and m5C-modified transcripts were involved in central metabolic pathways related to the life cycle, with modifications on the target genes selected in a tissue-specific manner. Furthermore, most modified sites were located within quantitative trait loci that control important agronomic traits, highlighting the value of cloning functional loci. The results provide new insights into the expression regulation complexity and data resource of the transcriptome and epitranscriptome, improving our understanding of the rice genome.

Genome-Wide Identification and Characterization of Heat Shock Protein 20 Genes in Maize.

Maize is an important cereal crop worldwide and is sensitive to abiotic stresses in fluctuant environments that seriously affect its growth, yield, and quality. The small heat shock protein (HSP20) plays a crucial role in protecting plants from abiotic stress. However, little is known about HSP20 in maize (ZmHSP20). In this study, 44 ZmHSP20s were identified, which were unequally distributed over 10 chromosomes, and 6 pairs of ZmHSP20s were tandemly presented. The gene structure of ZmHSP20s was highly conserved, with 95% (42) of the genes having no more than one intron. The analysis of the cis-element in ZmHSP20s promoter demonstrated large amounts of elements related to hormonal and abiotic stress responses, including abscisic acid (ABA), high temperature, and hypoxia. The ZmHSP20s protein had more than two conserved motifs that were predictably localized in the cytoplasm, nucleus, endoplasmic reticulum, peroxisome, mitochondria, and plasma. Phylogenetic analysis using HSP20s in Arabidopsis, rice, maize, and Solanum tuberosum indicated that ZmHSP20s were classified into 11 categories, of which each category had unique subcellular localization. Approximately 80% (35) of ZmHSP20 were upregulated under heat stress at the maize seedling stage, whereas the opposite expression profiling of 10 genes under 37 and 48 °C was detected. A total of 20 genes were randomly selected to investigate their expression under treatments of ABA, gibberellin (GA), ethylene, low temperature, drought, and waterlogging, and the results displayed that more than half of these genes were downregulated while ZmHSP20-3, ZmHSP20-7, ZmHSP20-24, and ZmHSP20-44 were upregulated under 1 h treatment of ethylene. A yeast-one-hybrid experiment was conducted to analyze the binding of four heat stress transcription factors (ZmHSFs) with eight of the ZmHSP20s promoter sequences, in which ZmHSF3, ZmHSF13, and ZmHSF17 can bind to most of these selected ZmHSP20s promoters. Our results provided a valuable resource for studying HSP20s function and offering candidates for genetic improvement under abiotic stress.

Jasmonic Acid-Involved OsEDS1 Signaling in Rice-Bacteria Interactions.

BACKGROUND: The function of Arabidopsis enhanced disease susceptibility 1 (AtEDS1) and its sequence homologs in other dicots have been extensively studied. However, it is unknown whether rice EDS1 homolog (OsEDS1) plays a role in regulating the rice-pathogen interaction. RESULTS: In this study, a OsEDS1-knouckout mutant (oseds1) was characterized and shown to have increased susceptibility to Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), suggesting the positive role of OsEDS1 in regulating rice disease resistance. However, the following evidence suggests that OsEDS1 shares some differences with AtEDS1 in its way to regulate the host-pathogen interactions. Firstly, OsEDS1 modulates the rice-bacteria interactions involving in jasmonic acid (JA) signaling pathway, while AtEDS1 regulates Arabidopsis disease resistance against biotrophic pathogens depending on salicylic acid (SA) signaling pathway. Secondly, introducing AtEDS1 could reduce oseds1 mutant susceptibility to Xoo rather than to Xoc. Thirdly, exogenous application of JA and SA cannot complement the susceptible phenotype of the oseds1 mutant, while exogenous application of SA is capable of complementing the susceptible phenotype of the ateds1 mutant. Finally, OsEDS1 is not required for R gene mediated resistance, while AtEDS1 is required for disease resistance mediated by TIR-NB-LRR class of R proteins. CONCLUSION: OsEDS1 is a positive regulator in rice-pathogen interactions, and shares both similarities and differences with AtEDS1 in its way to regulate plant-pathogen interactions.

Overexpression a "fruit-weight 2.2-like" gene OsFWL5 improves rice resistance.

BACKGROUND: Rice (Oryza sativa) feeds half of the world's population. Rice grain yield and quality which are constrained by diseases and mineral nutritions have important human healthy impacts. Plant "fruit-weight 2.2-like" (FWL) genes play key roles in modulating plant fruit weight, organ size and iron distribution. Previous work has uncovered that the grains of OsFWL5-oeverexpressing rice accumulated more beneficial element zinc (Zn) and less toxic element cadmium (Cd) content. However, whether FWL genes play roles in rice resistance remains unknown. FINDINGS: Here, we validated that one of rice FWL genes OsFWL5 plays a positive role in defense to Xanthomonas oryzae pv. oryzae (Xoo). Overexpresion of OsFWL5 promotes H2O2 accumulation and cell death. The OsFWL5-overexpresing plants show activated flg22-induced reactive oxygen species (ROS) generation, and increased resistance to Xoo, indicating that OsFWL5 functions to increase pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. The activated defense response is associated with increased the expression of genes involved in jasmonic acid (JA)-related signaling. Furthermore, Cd can induce rice resistance to Xoo, and OsFWL5 is required for Cd-induced rice defense response. CONCLUSION: Putting our finds and previous work together, OsFWL5 could be a candiate gene for breeders to genetically improve rice resistance and grain quality.

Xanthomonas oryzae pv. oryzae Inoculation and Growth Rate on Rice by Leaf Clipping Method.

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious bacterial diseases and a major impediment to the increase of rice yield. Appropriate methods for inoculation of Xoo and disease scoring are necessary to investigate the nature of the disease and the mechanism of plant resistance to the pathogen. As the most-widely grown crop in the worldwide, rice yield plays an important role in food security. Uncovering mechanisms of plant-pathogen interaction of rice and Xoo will help develop rice plants that are more resistant to disease caused by Xoo. Here we describe our validated and efficient methods for inoculation of Xoo and disease scoring.

Improvement of multiple agronomic traits by a disease resistance gene via cell wall reinforcement.

The major disease resistance gene Xa4 confers race-specific durable resistance against Xanthomonas oryzae pv. oryzae, which causes the most damaging bacterial disease in rice worldwide. Although Xa4 has been one of the most widely exploited resistance genes in rice production worldwide, its molecular nature remains unknown. Here we show that Xa4, encoding a cell wall-associated kinase, improves multiple traits of agronomic importance without compromising grain yield by strengthening the cell wall via promoting cellulose synthesis and suppressing cell wall loosening. Strengthening of the cell wall by Xa4 enhances resistance to bacterial infection, and also increases mechanical strength of the culm with slightly reduced plant height, which may improve lodging resistance of the rice plant. The simultaneous improvement of multiple agronomic traits conferred by Xa4 may account for its widespread and lasting utilization in rice breeding programmes globally.

A host basal transcription factor is a key component for infection of rice by TALE-carrying bacteria.

Transcription activator-like effectors (TALEs) are sequence-specific DNA binding proteins found in a range of plant pathogenic bacteria, where they play important roles in host-pathogen interactions. However, it has been unclear how TALEs, after they have been injected into the host cells, activate transcription of host genes required for infection success. Here, we show that the basal transcription factor IIA gamma subunit TFIIAγ5 from rice is a key component for infection by the TALE-carrying bacterium Xanthomonas oryzae pv. oryzae, the causal agent for bacterial blight. Direct interaction of several TALEs with TFIIAγ5 is required for activation of disease susceptibility genes. Conversely, reduced expression of the TFIIAγ5 host gene limits the induction of susceptibility genes and thus decreases bacterial blight symptoms. Suppression or mutation of TFIIAγ5 can also reduce bacterial streak, another devastating disease of rice caused by TALE-carrying X. oryzae pv. oryzicola. These results have important implications for formulating a widely applicable strategy with which to improve resistance of plants to TALE-carrying pathogens.