Osa-miR160a confers broad-spectrum resistance to fungal and bacterial pathogens in rice.

Rice production is threatened by multiple pathogens. Breeding cultivars with broad-spectrum disease resistance is necessary to maintain and improve crop production. Previously we found that overexpression of miR160a enhanced rice blast disease resistance. However, it is unclear whether miR160a also regulates resistance against other pathogens, and what the downstream signaling pathways are. Here, we demonstrate that miR160a positively regulates broad-spectrum resistance against the causative agents of blast, leaf blight and sheath blight in rice. Mutations of miR160a-targeted Auxin Response Factors result in different alteration of resistance conferred by miR160a. miR160a enhances disease resistance partially by suppressing ARF8, as mutation of ARF8 in MIM160 background partially restores the compromised resistance resulting from MIM160. ARF8 protein binds directly to the promoter and suppresses the expression of WRKY45, which acts as a positive regulator of rice immunity. Mutation of WRKY45 compromises the enhanced blast resistance and bacterial leaf blight resistance conferred by arf8 mutant. Overall, our results reveal that a microRNA coordinates rice broad-spectrum disease resistance by suppressing multiple target genes that play different roles in disease resistance, and uncover a new regulatory pathway mediated by the miR160a-ARF8 module. These findings provide new resources to potentially improve disease resistance for breeding in rice.

Molecular analysis of nematode-responsive defence genes CRF1, WRKY45, and PR7 in Solanum lycopersicum tissues during the infection of plant-parasitic nematode species of the genus Meloidogyne.

Several pathogens, including nematodes, have severe effects on plant development and growth, and immense populations of parasitic nematodes may cause plant death and crop loss. Obligate plant-parasitic nematodes and root-knot nematodes belonging to the genus Meloidogyne are significant parasites in crops. During nematode infection, damage-associated molecular patterns play a role in the activation of plant defence responses to pathogens. Several genes are involved in Meloidogyne parasitism. However, the expression of nematode-responsive genes CRF1, WRKY45, and PR7 during infection with different parasitic nematode species is not well understood. Therefore, this study aimed to reveal plant responses to differential gene expression of nematode-responsive genes in tomato plants, and their relationship to nematode reproduction and comparative phylogeny. Molecular methods for gene expression, greenhouse work for nematode reproduction, and phylogenetic analysis were used to determine nematode-plant interactions. The results revealed that differential gene expression of CRF1, WRKY45, and PR7 depended on the nematode species. The relative CRF1 gene expression reached its highest level at 3 dpi, following nematode infection. In conclusion, plant defense responses disturbed the expression of nematode-responsive genes, and the differential expression of nematode-responsive genes was affected by nematode species and nematode parasitism.

Mitochondrial signalling is critical for acclimation and adaptation to flooding in Arabidopsis thaliana.

Mitochondria have critical functions in the acclimation to abiotic and biotic stresses. Adverse environmental conditions lead to increased demands in energy supply and metabolic intermediates, which are provided by mitochondrial ATP production and the tricarboxylic acid (TCA) cycle. Mitochondria also play a role as stress sensors to adjust nuclear gene expression via retrograde signalling with the transcription factor (TF) ANAC017 and the kinase CDKE1 key components to integrate various signals into this pathway. To determine the importance of mitochondria as sensors of stress and their contribution in the tolerance to adverse growth conditions, a comparative phenotypical, physiological and transcriptomic characterisation of Arabidopsis mitochondrial signalling mutants (cdke1/rao1 and anac017/rao2) and a set of contrasting accessions was performed after applying the complex compound stress of submergence. Our results showed that impaired mitochondrial retrograde signalling leads to increased sensitivity to the stress treatments. The multi-factorial approach identified a network of 702 co-expressed genes, including several WRKY TFs, overlapping in the transcriptional responses in the mitochondrial signalling mutants and stress-sensitive accessions. Functional characterisation of two WRKY TFs (WRKY40 and WRKY45), using both knockout and overexpressing lines, confirmed their role in conferring tolerance to submergence. Together, the results revealed that acclimation to submergence is dependent on mitochondrial retrograde signalling, and underlying transcriptional re-programming is used as an adaptation mechanism.

Arabidopsis transcription factor WRKY45 confers cadmium tolerance via activating PCS1 and PCS2 expression.

Cadmium (Cd) has long been recognized as toxic pollutant to crops worldwide. The biosynthesis of glutathione-dependent phytochelatin (PC) plays crucial roles in the detoxification of Cd in plants. However, its regulatory mechanism remains elusive. Here, we revealed that Arabidopsis transcription factor WRKY45 confers Cd tolerance via promoting the expression of PC synthesis-related genes PCS1 and PCS2, respectively. Firstly, we found that Cd stress induces the transcript levels of WRKY45 and its protein abundance. Accordingly, in contrast to wild type Col-0, the increased sensitivity to Cd is observed in wrky45 mutant, while overexpressing WRKY45 plants are more tolerant to Cd. Secondly, quantitative real-time PCR revealed that the expression of AtPCS1 and AtPCS2 is stimulated in overexpressing WRKY45 plants, but decreased in wrky45 mutant. Thirdly, WRKY45 promotes the expression of PCS1 and PCS2, electrophoresis mobility shift assay analysis uncovered that WRKY45 directly binds to the W-box cis-element of PCS2 promoter. Lastly, the overexpression of WRKY45 in Col-0 leads to more accumulation of PCs in Arabidopsis, and the overexpression of PCS1 or PCS2 in wrky45 mutant plants rescues the phenotypes induced by Cd stress. In conclusion, our results show that AtWRKY45 positively regulates Cd tolerance in Arabidopsis via activating PCS1 and PCS2 expression.

Rice ubiquitin-conjugating enzyme OsUBC26 is essential for immunity to the blast fungus Magnaporthe oryzae.

The functions of ubiquitin-conjugating enzymes (E2) in plant immunity are not well understood. In this study, OsUBC26, a rice ubiquitin-conjugating enzyme, was characterized in the defence against Magnaporthe oryzae. The expression of OsUBC26 was induced by M. oryzae inoculation and methyl jasmonate treatment. Both RNA interference lines and CRISPR/Cas9 null mutants of OsUBC26 reduced rice resistance to M. oryzae. WRKY45 was down-regulated in OsUBC26 null mutants. In vitro E2 activity assay indicated that OsUBC26 is an active ubiquitin-conjugating enzyme. Yeast two-hybrid assays using OsUBC26 as bait identified the RING-type E3 ligase UCIP2 as an interacting protein. Coimmunoprecipitation assays confirmed the interaction between OsUBC26 and UCIP2. The CRISPR/Cas9 mutants of UCIP2 also showed compromised resistance to M. oryzae. Yeast two-hybrid screening using UCIP2 as bait revealed that APIP6 is a binding partner of UCIP2. Moreover, OsUBC26 working with APIP6 ubiquitinateds AvrPiz-t, an avirulence effector of M. oryzae, and OsUBC26 null mutation impaired the proteasome degradation of AvrPiz-t in rice cells. In summary, OsUBC26 plays important roles in rice disease resistance by regulating WRKY45 expression and working with E3 ligases such as APIP6 to counteract the effector protein AvrPiz-t from M. oryzae.

Integrative Metabolomic and Transcriptomic Analyses Reveal Metabolic Changes and Its Molecular Basis in Rice Mutants of the Strigolactone Pathway.

Plants have evolved many metabolites to meet the demands of growth and adaptation. Although strigolactones (SLs) play vital roles in controlling plant architecture, their function in regulating plant metabolism remains elusive. Here we report the integrative metabolomic and transcriptomic analyses of two rice SL mutants, d10 (a biosynthesis mutant) and d14 (a perception mutant). Both mutants displayed a series of metabolic and transcriptional alterations, especially in the lipid, flavonoid, and terpenoid pathways. Levels of several diterpenoid phytoalexins were substantially increased in d10 and d14, together with the induction of terpenoid gene cluster and the corresponding upstream transcription factor WRKY45, an established determinant of plant immunity. The fact that WRKY45 is a target of IPA1, which acted as a downstream transcription factor of SL signaling, suggests that SLs contribute to plant defense through WRKY45 and phytoalexins. Moreover, our data indicated that SLs may modulate rice metabolism through a vast number of clustered or tandemly duplicated genes. Our work revealed a central role of SLs in rice metabolism. Meanwhile, integrative analysis of the metabolome and transcriptome also suggested that SLs may contribute to metabolite-associated growth and defense.

Expressing a Target Mimic of miR156fhl-3p Enhances Rice Blast Disease Resistance Without Yield Penalty by Improving SPL14 Expression.

MicroRNAs (miRNAs) play essential roles in the regulation of plant growth and defense responses. More and more, miRNA-3ps are reported to act in plant development and immunity. miR156 is a conserved miRNA, and most previous studies focus on its roles in plant growth, development, and yield determinacy. Here, we show that expressing a target mimic of miR156fhl-3p led to enhanced rice blast disease resistance without a yield penalty. miR156fhl-3p was differentially responsive to Magnaporthe oryzae in susceptible and resistant accessions. Transgenic lines expressing a target mimic of miR156fhl-3p (MIM156-3p) exhibited enhanced rice blast disease resistance and increased expression of defense-related genes. MIM156-3p also enhanced the mRNA abundance of SPL14 and WRKY45 by down-regulating miR156-5p and pre-miR156. Moreover, MIM156-3p lines displayed a decreased number of second rachis branches per panicle but enlarged grains, leading to unchanged yield per plant. Consistently, overexpressing miR156h (OX156) led to enhanced susceptibility to M. oryzae and decreased the expression of SPL14 and WRKY45. Our results indicate that miR156fhl-3p mounts a regulatory role on miR156-5p, which subsequently regulates the expression of SPL14 and WRKY45 to improve rice blast disease resistance.

WRKY45 phosphorylation at threonine 266 acts negatively on WRKY45-dependent blast resistance in rice.

WRKY45 is a central regulator of disease resistance mediated by salicylic acid signaling in rice and its activation involves phosphorylation by OsMPK6. OsMPK6 phosphorylates WRKY45 at Thr266, Ser294, and Ser299 in vitro. Phosphorylation of Ser294 and/or Ser299 is required for full activation of WRKY45, but the importance of Thr266 phosphorylation has remained unknown. Here, we report on the characterization of Thr266 phosphorylation of WRKY45 in rice. Transient expression of mutant WRKY45 revealed that Thr266 is phosphorylated in vivo, together with Ser294/299. Replacement of Thr266 by Asn did not affect the enhanced Magnaporthe oryzae resistance afforded by WRKY45 overexpression. By contrast, replacement by Asp negated the enhancement of M. oryzae resistance. These results suggest that Thr266 phosphorylation acts negatively on WRKY45-dependent disease resistance.

Regulating Tradeoffs to Improve Rice Production.

Plants are sessile organisms that are continuously exposed to a wide range of environmental stresses. To cope with various stresses using limited resources, plants have evolved diverse mechanisms of "tradeoff" that enable the allocation of resources to address the most life-threatening stress. During our studies on induced disease resistance in rice, we have found some important phenomena relevant to tradeoffs between biotic and abiotic stress responses, and between stress response and plant growth. We characterized these tradeoff phenomena from viewpoints of signaling crosstalks associated with transcriptional regulation. Here, I describe following topics: (1) PTP1-dependent increased disease susceptibility of rice under low temperature and high salinity conditions, (2) OsNPR1-dependent tradeoff between pathogen defense and photosynthesis, (3) tradeoff between pathogen defense and abiotic stress tolerance in WRKY45-overexpressing rice plants, and (4) WRKY62-dependent tradeoff between pathogen defense and hypoxia tolerance. Lastly, I discuss my view regarding the significance of such tradeoffs in agricultural production that should be considered in crop breeding; that is, the tradeoffs, although they benefit plants in nature, can be rather disadvantageous in agricultural production.

Development of disease-resistant rice using regulatory components of induced disease resistance.

Infectious diseases cause huge crop losses annually. In response to pathogen attacks, plants activate defense systems that are mediated through various signaling pathways. The salicylic acid (SA) signaling pathway is the most powerful of these pathways. Several regulatory components of the SA signaling pathway have been identified, and are potential targets for genetic manipulation of plants' disease resistance. However, the resistance associated with these regulatory components is often accompanied by fitness costs; that is, negative effects on plant growth and crop yield. Chemical defense inducers, such as benzothiadiazole and probenazole, act on the SA pathway and induce strong resistance to various pathogens without major fitness costs, owing to their 'priming effect.' Studies on how benzothiadiazole induces disease resistance in rice have identified WRKY45, a key transcription factor in the branched SA pathway, and OsNPR1/NH1. Rice plants overexpressing WRKY45 were extremely resistant to rice blast disease caused by the fungus Magnaporthe oryzae and bacterial leaf blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo), the two major rice diseases. Disease resistance is often accompanied by fitness costs; however, WRKY45 overexpression imposed relatively small fitness costs on rice because of its priming effect. This priming effect was similar to that of chemical defense inducers, although the fitness costs were amplified by some environmental factors. WRKY45 is degraded by the ubiquitin-proteasome system, and the dual role of this degradation partly explains the priming effect. The synergistic interaction between SA and cytokinin signaling that activates WRKY45 also likely contributes to the priming effect. With a main focus on these studies, I review the current knowledge of SA-pathway-dependent defense in rice by comparing it with that in Arabidopsis, and discuss potential strategies to develop disease-resistant rice using signaling components.

Reduced expression of glycolate oxidase leads to enhanced disease resistance in rice.

Glycolate oxidase (GLO) is a key enzyme in photorespiration, catalyzing the oxidation of glycolate to glyoxylate. Arabidopsis GLO is required for nonhost defense responses to Pseudomonas syringae and for tobacco Pto/AvrPto-mediated defense responses. We previously described identification of rice GLO1 that interacts with a glutaredoxin protein, which in turn interacts with TGA transcription factors. TGA transcription factors are well known to participate in NPR1/NH1-mediated defense signaling, which is crucial to systemic acquired resistance in plants. Here we demonstrate that reduction of rice GLO1 expression leads to enhanced resistance to Xanthomonas oryzae pv oryzae (Xoo). Constitutive silencing of GLO1 leads to programmed cell death, resulting in a lesion-mimic phenotype and lethality or reduced plant growth and development, consistent with previous reports. Inducible silencing of GLO1, employing a dexamethasone-GVG (Gal4 DNA binding domain-VP16 activation domain-glucocorticoid receptor fusion) inducible system, alleviates these detrimental effects. Silencing of GLO1 results in enhanced resistance to Xoo, increased expression of defense regulators NH1, NH3, and WRKY45, and activation of PR1 expression.