Physiological and transcriptomic analyses reveal novel insights into the cultivar-specific response to alkaline stress in alfalfa (Medicago sativa L.).

Soil alkalization severely limits plant growth and development, however, the mechanisms of alkaline response in plants remain largely unknown. In this study, we performed physiological and transcriptomic analyses using two alfalfa cultivars (Medicago sativa L.) with different sensitivities to alkaline conditions. The chlorophyll content and shoot fresh mass drastically declined in the alkaline-sensitive cultivar Algonquin (AG) following alkaline treatment (0-25 mM Na2CO3 solution), while the alkaline-tolerant cultivar Gongnong NO.1 (GN) maintained relatively stable growth and chlorophyll content. Compared with AG, GN had higher contents of Ca2+ and Mg2+; the ratios of Ca2+ and Mg2+ to Na+, proline and soluble sugar, as well as higher enzyme activities of peroxidase (POD) and catalase (CAT) under the alkaline conditions. Furthermore, transcriptomic analysis identified three categories of alkaline-responsive differentially expressed genes (DEGs) between the two cultivars: 48 genes commonly induced in both the cultivars (CAR), 574 genes from the tolerant cultivar (TAR), and 493 genes from the sensitive cultivar (SAR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that CAR genes were mostly involved in phenylpropanoid biosynthesis, lipid metabolism, and DNA replication and repair; TAR genes were significantly enriched in metabolic pathways, such as biosynthesis of amino acids and secondary metabolites including flavonoids, and the MAPK signaling pathway; SAR genes were specifically enriched in vitamin B6 metabolism. Taken together, the results identified candidate pathways associated with genetic variation in response to alkaline stress, providing novel insights into the mechanisms underlying alkaline tolerance in alfalfa.

A fresh weight-based method for evaluating soybean resistance to red crown rot.

Soybean red crown rot (RCR) caused by Calonectria ilicicola is a serious soil-borne disease affecting soybean production and quality. The current visual necrosis-based method for the measurement of RCR severity is prone to subjectivity as well as time consuming and laborious as it requires digging out and washing the roots to remove adhering soil prior to the visual scoring. Using cultivar Enrei, we show that, upon C. ilicicola infection, relative fresh weights (RFW; fresh weights relative to non-inoculated control plants) showed a significant negative correlation with visual RCR severity in apical shoot (trifoliate and above, R2 = 0.96), shoot (unifoliate and above, R2 = 0.82) and roots (R2 = 0.89). Furthermore, apical shoot RFW efficiently correlated with varying levels of C. ilicicola resistance in two test sets containing 37 soybean cultivars and three wild soybean accessions, exhibiting a significant correlation with visual severity (R2 = 0.72 and 0.79, p < 0.01). Taken together, our results suggest that RFW can serve as an index of soybean RCR severity, providing a simple, rapid, consistent, and cost-effective method for evaluating C. ilicicola resistance in soybeans.

Rice OsAAA-ATPase1 is Induced during Blast Infection in a Salicylic Acid-Dependent Manner, and Promotes Blast Fungus Resistance.

Fatty acids (FAs) have been implicated in signaling roles in plant defense responses. We previously reported that mutation or RNAi-knockdown (OsSSI2-kd) of the rice OsSSI2 gene, encoding a stearoyl acyl carrier protein FA desaturase (SACPD), remarkably enhanced resistance to blast fungus Magnaporthe oryzae and the leaf-blight bacterium Xanthomonas oryzae pv. oryzae (Xoo). Transcriptomic analysis identified six AAA-ATPase family genes (hereafter OsAAA-ATPase1-6) upregulated in the OsSSI2-kd plants, in addition to other well-known defense-related genes. Here, we report the functional analysis of OsAAA-ATPase1 in rice's defense response to M. oryzae. Recombinant OsAAA-ATPase1 synthesized in Escherichia coli showed ATPase activity. OsAAA-ATPase1 transcription was induced by exogenous treatment with a functional analogue of salicylic acid (SA), benzothiadiazole (BTH), but not by other plant hormones tested. The transcription of OsAAA-ATPase1 was also highly induced in response to M. oryzae infection in an SA-dependent manner, as gene induction was significantly attenuated in a transgenic rice line expressing a bacterial gene (nahG) encoding salicylate hydroxylase. Overexpression of OsAAA-ATPase1 significantly enhanced pathogenesis-related gene expression and the resistance to M. oryzae; conversely, RNAi-mediated suppression of this gene compromised this resistance. These results suggest that OsAAA-APTase1 plays an important role in SA-mediated defense responses against blast fungus M. oryzae.

Tyrosine phosphorylation of a receptor-like cytoplasmic kinase, BSR1, plays a crucial role in resistance to multiple pathogens in rice.

Plants have evolved many receptor-like cytoplasmic kinases (RLCKs) to modulate their growth, development, and innate immunity. Broad-Spectrum Resistance 1 (BSR1) encodes a rice RLCK, whose overexpression confers resistance to multiple diseases, including fungal rice blast and bacterial leaf blight. However, the mechanisms underlying resistance remain largely unknown. In the present study, we report that BSR1 is a functional protein kinase that autophosphorylates and transphosphorylates an artificial substrate in vitro. Although BSR1 is classified as a serine/threonine kinase, it was shown to autophosphorylate on tyrosine as well as on serine/threonine residues when expressed in bacteria, demonstrating that it is a dual-specificity kinase. Protein kinase activity was found to be indispensable for resistance to rice blast and leaf blight in BSR1-overexpressing plants. Importantly, tyrosine phosphorylation of BSR1 was critical for proper localization of BSR1 in rice cells and played a crucial role in BSR1-mediated resistance to multiple diseases, as evidenced by compromised disease resistance in transgenic plants overexpressing a mutant BSR1 in which Tyr-63 was substituted with Ala. Overall, our data indicate that BSR1 is a non-receptor dual-specificity kinase and that both tyrosine and serine/threonine kinase activities are critical for the normal functioning of BSR1 in the resistance to multiple pathogens. Our results support the notion that tyrosine phosphorylation plays a major regulatory role in the transduction of defense signals from cell-surface receptor complexes to downstream signaling components in plants.

Abiotic Stresses Antagonize the Rice Defence Pathway through the Tyrosine-Dephosphorylation of OsMPK6.

Plants, as sessile organisms, survive environmental changes by prioritizing their responses to the most life-threatening stress by allocating limited resources. Previous studies showed that pathogen resistance was suppressed under abiotic stresses. Here, we show the mechanism underlying this phenomenon. Phosphorylation of WRKY45, the central transcription factor in salicylic-acid (SA)-signalling-dependent pathogen defence in rice, via the OsMKK10-2-OsMPK6 cascade, was required to fully activate WRKY45. The activation of WRKY45 by benzothiadiazole (BTH) was reduced under low temperature and high salinity, probably through abscisic acid (ABA) signalling. An ABA treatment dephosphorylated/inactivated OsMPK6 via protein tyrosine phosphatases, OsPTP1/2, leading to the impaired activation of WRKY45 and a reduction in Magnaporthe oryzae resistance, even after BTH treatment. BTH induced a strong M. oryzae resistance in OsPTP1/2 knockdown rice, even under cold and high salinity, indicating that OsPTP1/2 is the node of SA-ABA signalling crosstalk and its down-regulation makes rice disease resistant, even under abiotic stresses. These results points to one of the directions to further improve crops by managing the tradeoffs between different stress responses of plants.

Evaluation of Resistance to Phytophthora sojae in Soybean Mini Core Collections Using an Improved Assay System.

Stem and root rot disease caused by Phytophthora sojae is devastating to soybean crops worldwide. Developing host resistance to P. sojae, considered the most effective and stable means to control this disease, is partly hampered by limited germplasm resources. In this study, we first modified conventional methods for a P. sojae resistance assay to a simpler and more cost-effective version, in which the P. sojae inoculum was mixed into the soil and the resistance was evaluated by survival rate (%) of soybean seedlings. This rating had significant correlations (P < 0.01) with the reduction in root fresh weight and the visual root rot severity. Applying this method to evaluate P. sojae resistance in soybean mini core collections comprising either 79 accessions originating from Japan (JMC) or 80 accessions collected around the world (WMC) revealed a wide variation in resistance among the individual varieties. In total, 38 accessions from the JMC and 41 from the WMC exhibited resistance or moderate resistance to P. sojae isolate N1 (with virulence to Rps1b, 3c, 4, 5, and 6), with ≥50% survival. Of these, 26 from the JMC and 29 from the WMC showed at least moderate resistance to P. sojae isolate HR1 (vir Rps1a-c, 1k, 2, 3a-c, 4-6, and 8). Additionally, 24 WCS accessions, in contrast to only 6 from the JMC, exhibited 100% survival after being challenged with both the N1 and HR1 isolates, suggesting a biogeographical difference between the two collections. We further verified two JMC varieties, Daizu and Amagi zairai 90D, for their resistance to an additional four P. sojae isolates (60 to 100% survival), which may provide new and valuable genetic sources for P. sojae resistance breeding in soybean.

Rice OsVAMP714, a membrane-trafficking protein localized to the chloroplast and vacuolar membrane, is involved in resistance to rice blast disease.

Membrane trafficking plays pivotal roles in many cellular processes including plant immunity. Here, we report the characterization of OsVAMP714, an intracellular SNARE protein, focusing on its role in resistance to rice blast disease caused by the fungal pathogen Magnaporthe oryzae. Disease resistance tests using OsVAMP714 knockdown and overexpressing rice plants demonstrated the involvement of OsVAMP714 in blast resistance. The overexpression of OsVAMP7111, whose product is highly homologous to OsVAMP714, did not enhance blast resistance to rice, implying a potential specificity of OsVAMP714 to blast resistance. OsVAMP714 was localized to the chloroplast in mesophyll cells and to the cellular periphery in epidermal cells of transgenic rice plant leaves. We showed that chloroplast localization is critical for the normal OsVAMP714 functioning in blast resistance by analyzing the rice plants overexpressing OsVAMP714 mutants whose products did not localize in the chloroplast. We also found that OsVAMP714 was located in the vacuolar membrane surrounding the invasive hyphae of M. oryzae. Furthermore, we showed that OsVAMP714 overexpression promotes leaf sheath elongation and that the first 19 amino acids, which are highly conserved between animal and plant VAMP7 proteins, are crucial for normal rice plant growths. Our studies imply that the OsVAMP714-mediated trafficking pathway plays an important role in rice blast resistance as well as in the vegetative growth of rice.

Blast resistance of CC-NB-LRR protein Pb1 is mediated by WRKY45 through protein-protein interaction.

Panicle blast 1 (Pb1) is a panicle blast resistance gene derived from the indica rice cultivar "Modan." Pb1 encodes a coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NB-LRR) protein and confers durable, broad-spectrum resistance to Magnaporthe oryzae races. Here, we investigated the molecular mechanisms underlying Pb1-mediated blast resistance. The Pb1 protein interacted with WRKY45, a transcription factor involved in induced resistance via the salicylic acid signaling pathway that is regulated by the ubiquitin proteasome system. Pb1-mediated panicle blast resistance was largely compromised when WRKY45 was knocked down in a Pb1-containing rice cultivar. Leaf-blast resistance by Pb1 overexpression (Pb1-ox) was also compromised in WRKY45 knockdown/Pb1-ox rice. Blast infection induced higher accumulation of WRKY45 in Pb1-ox than in control Nipponbare rice. Overexpression of Pb1-Quad, a coiled-coil domain mutant that had weak interaction with WRKY45, resulted in significantly weaker blast resistance than that of wild-type Pb1. Overexpression of Pb1 with a nuclear export sequence failed to confer blast resistance to rice. These results suggest that the blast resistance of Pb1 depends on its interaction with WRKY45 in the nucleus. In a transient system using rice protoplasts, coexpression of Pb1 enhanced WRKY45 accumulation and increased WRKY45-dependent transactivation activity, suggesting that protection of WRKY45 from ubiquitin proteasome system degradation is possibly involved in Pb1-dependent blast resistance.

WRKY45-dependent priming of diterpenoid phytoalexin biosynthesis in rice and the role of cytokinin in triggering the reaction.

Plant activators such as benzothiadiazole (BTH) protect plants against diseases by priming the salicylic acid (SA) signaling pathway. In rice, the transcription factor WRKY45 plays a central role in this process. To investigate the mechanism involved in defense-priming by BTH and the role of WRKY45 in this process, we analyzed the transcripts of biosynthetic genes for diterpenoid phytoalexins (DPs) during the rice-Magnaporthe oryzae interaction. The DP biosynthetic genes were barely upregulated in BTH-treated rice plants, but were induced rapidly after M. oryzae infection in a WRKY45-dependent manner. These results indicate that the DP biosynthetic genes were primed by BTH through WRKY45. Rapid induction of the DP biosynthetic genes was also observed after M. oryzae infection to WRKY45-overexpressing (WRKY45-ox) plants. The changes in gene transcription resulted in accumulation of DPs in WRKY45-ox and BTH-pretreated rice after M. oryzae infection. Previously, we reported that cytokinins (CKs), especially isopentenyladenines, accumulated in M. oryzae-infected rice. Here, we show that DP biosynthetic genes are regulated by the SA/CK synergism in a WRKY45-dependent manner. Together, we propose that CK plays a role in mediating the signal of M. oryzae infection to trigger the induction of DP biosynthetic genes in BTH-primed plants.

Antioxidant Agriculture for Stress-Resilient Crop Production: Field Practice.

Oxidative stress, resulting from the excessive production of reactive oxygen species, is a common and major cause of cellular damage in plants exposed to various abiotic stresses. To address this challenge, we introduce the concept of antioxidant agriculture as a comprehensive strategy to improve stress tolerance and thus crop productivity by minimizing oxidative stress levels in the field environment. This strategy encompasses a diverse range of approaches, including genetic engineering, the exogenous application of antioxidant agents, microbial inoculation, and agronomic practices, to reinforce the plant's intrinsic antioxidant defense system and mitigate oxidative stress. We present recent successful studies of antioxidant measures that have been validated in field conditions, along with our perspective on achieving antioxidant agriculture.

Cytokinins act synergistically with salicylic acid to activate defense gene expression in rice.

Hormone crosstalk is pivotal in plant-pathogen interactions. Here, we report on the accumulation of cytokinins (CK) in rice seedlings after infection of blast fungus Magnaporthe oryzae and its potential significance in rice-M. oryzae interaction. Blast infection to rice seedlings increased levels of N(6)-(Δ(2)-isopentenyl) adenine (iP), iP riboside (iPR), and iPR 5'-phosphates (iPRP) in leaf blades. Consistent with this, CK signaling was activated around the infection sites, as shown by histochemical staining for ß-glucuronidase activity driven by a CK-responsive OsRR6 promoter. Diverse CK species were also detected in the hyphae (mycelium), conidia, and culture filtrates of blast fungus, indicating that M. oryzae is capable of production as well as hyphal secretion of CK. Co-treatment of leaf blades with CK and salicylic acid (SA), but not with either one alone, markedly induced pathogenesis-related genes OsPR1b and probenazole-induced protein 1 (PBZ1). These effects were diminished by RNAi-knockdown of OsNPR1 or WRKY45, the key regulators of the SA signaling pathway in rice, indicating that the effects of CK depend on these two regulators. Taken together, our data imply a coevolutionary rice-M. oryzae interaction, wherein M. oryzae probably elevates rice CK levels for its own benefits such as nutrient translocation. Rice plants, on the other hand, sense it as an infection signal and activate defense reactions through the synergistic action with SA.

Genome-wide identification of WRKY45-regulated genes that mediate benzothiadiazole-induced defense responses in rice.

BACKGROUND: The rice transcription factor WRKY45 plays a crucial role in salicylic acid (SA)/benzothiadiazole (BTH)-induced disease resistance. Its knockdown severely reduces BTH-induced resistance to the fungal pathogen Magnaporthe oryzae and the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). Conversely, overexpression of WRKY45 induces extremely strong resistance to both of these pathogens. To elucidate the molecular basis of WRKY45-dependent disease resistance, we analyzed WRKY45-regulated gene expression using rice transformants and a transient gene expression system. RESULTS: We conducted a microarray analysis using WRKY45-knockdown (WRKY45-kd) rice plants, and identified WRKY45-dependent genes among the BTH-responsive genes. The BTH-responsiveness of 260 genes was dependent on WRKY45. Among these, 220 genes (85%), many of which encoded PR proteins and proteins associated with secondary metabolism, were upregulated by BTH. Only a small portion of these genes overlapped with those regulated by OsNPR1/NH1, supporting the idea that the rice SA pathway branches into WRKY45- regulated and OsNPR1/NH1-regulated subpathways. Dexamethazone-induced expression of myc-tagged WRKY45 in rice immediately upregulated transcription of endogenous WRKY45 and genes encoding the transcription factors WRKY62, OsNAC4, and HSF1, all of which have been reported to have defense-related functions. This was followed by upregulation of defense genes encoding PR proteins and secondary metabolic enzymes. Many of these genes were also induced after M. oryzae infection. Their temporal transcription patterns were consistent with those after dexamethazone-induced WRKY45 expression. In a transient expression system consisting of particle bombardment of rice coleoptiles, WRKY45 acted as an effector to trans-activate reporter genes in which the luciferase coding sequence was fused to upstream and intragenic sequences of WRKY62 and OsNAC4. Trans-activation of transcription occurred through a W-box-containing sequence upstream of OsNAC4 and mutations in the W-boxes abolished the trans-activation. CONCLUSIONS: These data suggest a role of WRKY45 in BTH-induced disease resistance as a master regulator of the transcriptional cascade regulating defense responses in one of two branches in the rice SA pathway.

Identification of Pseudomonas strains for the biological control of soybean red crown root rot.

Soybean red crown root rot (RCR), caused by the soil-borne fungal pathogen, Calonectria ilicicola, is the most destructive disease affecting soybean production in Japan. To date, no resistant cultivars or effective fungicides have been developed to control this disease. In this study, we evaluated 13 bacterial strains to determine their efficacy in controlling C. ilicicola. We first investigated whether the volatile organic compounds (VOCs) emitted by the bacterial strains exhibited any antifungal activity against C. ilicicola using the double-plate chamber method. The results showed that VOCs from three Pseudomonas bacterial strains, OFT2 (Pseudomonas sp.), OFT5 (Pseudomonas sp.), and Cab57 (Pseudomonas protegens), exhibited strong inhibitory activity against C. ilicicola mycelial growth. Some antifungal activity was also observed in the culture supernatants of these Pseudomonas strains. Greenhouse soil inoculation tests showed that application of OFT2, OFT5, and Cab57 cultures around soybean seeds after seed sowing significantly reduced the severity of RCR, as shown by up to 40% reduction in C. ilicicola fungal growth in the roots and 180-200% increase in shoot and root fresh weights compared to the water control. Our results suggest that OFT2, Cab57, and OFT5 produce potent antifungal compounds against C. ilicicola, thereby showing considerable potential for the biological control of C. ilicicola during soybean production.

Isolation, fine mapping and expression profiling of a lesion mimic genotype, spl(NF4050-8) that confers blast resistance in rice.

We evaluated a large collection of Tos17 mutant panel lines for their reaction to three different races of Magnaporthe oryzae and identified a lesion mimic mutant, NF4050-8, that showed lesions similar to naturally occurring spl5 mutant and enhanced resistance to all the three blast races tested. Nested modified-AFLP using Tos17-specific primers and southern hybridization experiments of segregating individuals indicated that the lesion mimic phenotype in NF4050-8 is most likely due to a nucleotide change acquired during the culturing process and not due to Tos17 insertion per se. Inheritance and genetic analyses in two japonica × indica populations identified an overlapping genomic region of 13 cM on short arm of chromosome 7 that was linked with the lesion mimic phenotype. High-resolution genetic mapping using 950 F(3) and 3,821 F(4) plants of NF4050-8 × CO39 delimited a 35 kb region flanked by NBARC1 (5.262 Mb) and RM8262 (5.297 Mb), which contained 6 ORFs; 3 of them were 'resistance gene related' with typical NBS-LRR signatures. One of them harbored a NB-ARC domain, which had been previously demonstrated to be associated with cell death in animals. Microarray analysis of NF4050-8 revealed significant up-regulation of numerous defense/pathogenesis-related genes and down-regulation of heme peroxidase genes. Real-time PCR analysis of WRKY45 and PR1b genes suggested possible constitutive activation of a defense signaling pathway downstream of salicylic acid but independent of NH1 in these mutant lines of rice.

Soybean Hypocotyls Prevent Calonectria ilicicola Invasion by Multi-Layered Defenses.

In plants, many pathogens infect a specific set of host organs to cause disease, yet the underlying mechanisms remain unclear. Here, we show that inoculation of soybean plants with Calonectria ilicicola, the soil-borne causal agent of soybean red crown rot, caused typical disease symptoms of root rot and leaf chlorosis and necrosis. However, the pathogen DNA was only detected in the roots and stem (hypocotyl) base but not other aerial parts of the plants. As we observed vigorous fungal growth in all culture media made of extracts from roots, stems, and leaves, differences in key components including available nutrients did not determine organ-specific infection and reproduction by C. ilicicola. Furthermore, inoculation of stems both with and without a surface wound showed that the stems resisted C. ilicicola infection via both the pre- and post-invasion defense layers. Transcriptomic comparison of roots and stems using RNA-seq analysis further revealed that upon C. ilicicola inoculation, a greater expression of genes involved in stress response was induced in the plant stems, including receptor-like kinase, AP2/ERF, MYB, and WRKY. In addition, pathways related to amino acid metabolism were also more upregulated in the stems in response to C. ilicicola infection. These results suggest that soybean stems provide C. ilicicola resistance, at least in part, by activating an organ-specific defense response.

Abscisic acid interacts antagonistically with salicylic acid signaling pathway in rice-Magnaporthe grisea interaction.

Plant hormones play pivotal signaling roles in plant-pathogen interactions. Here, we report characterization of an antagonistic interaction of abscisic acid (ABA) with salicylic acid (SA) signaling pathways in the rice-Magnaporthe grisea interaction. Exogenous application of ABA drastically compromised the rice resistance to both compatible and incompatible M. grisea strains, indicating that ABA negatively regulates both basal and resistance gene-mediated blast resistance. ABA markedly suppressed the transcriptional upregulation of WRKY45 and OsNPR1, the two key components of the SA signaling pathway in rice, induced by SA or benzothiadiazole or by blast infection. Overexpression of OsNPR1 or WRKY45 largely negated the enhancement of blast susceptibility by ABA, suggesting that ABA acts upstream of WRKY45 and OsNPR1 in the rice SA pathway. ABA-responsive genes were induced during blast infection in a pattern reciprocal to those of WRKY45 and OsPR1b in the compatible rice-blast interaction but only marginally in the incompatible one. These results suggest that the balance of SA and ABA signaling is an important determinant for the outcome of the rice-M. grisea interaction. ABA was detected in hyphae and conidia of M. grisea as well as in culture media, implying that blast-fungus-derived ABA could play a role in triggering ABA signaling at host infection sites.

Role of OsNPR1 in rice defense program as revealed by genome-wide expression analysis.

NPR1 is a central regulator of salicylic-acid (SA)-mediated defense signaling in Arabidopsis. Here, we report the characterization of OsNPR1, an Oryzae sativa (rice) ortholog of NPR1, focusing on its role in blast disease resistance and identification of OsNPR1-regulated genes. Blast resistance tests using OsNPR1 knockdown and overexpressing rice lines demonstrated the essential role of OsNPR1 in benzothiadiazole (BTH)-induced blast resistance. Genome-wide transcript profiling using OsNPR1-knockdown lines revealed that 358 genes out of 1,228 BTH-upregulated genes and 724 genes out of 1,069 BTH-downregulated genes were OsNPR1-dependent with respect to BTH responsiveness, thereby indicating that OsNPR1 plays a more vital role in gene downregulation. The OsNPR1-dependently downregulated genes included many of those involved in photosynthesis and in chloroplast translation and transcription. Reduction of photosynthetic activity after BTH treatment and its negation by OsNPR1 knockdown were indeed reflected in the changes in Fv/Fm values in leaves. These results imply the role of OsNPR1 in the reallocation of energy and resources during defense responses. We also examined the OsNPR1-dependence of SA-mediated suppression of ABA-induced genes.

Suppression of the rice fatty-acid desaturase gene OsSSI2 enhances resistance to blast and leaf blight diseases in rice.

Fatty acids and their derivatives play important signaling roles in plant defense responses. It has been shown that suppressing a gene for stearoyl acyl carrier protein fatty-acid desaturase (SACPD) enhances the resistance of Arabidopsis (SSI2) and soybean to multiple pathogens. In this study, we present functional analyses of a rice homolog of SSI2 (OsSSI2) in disease resistance of rice plants. A transposon insertion mutation (Osssi2-Tos17) and RNAi-mediated knockdown of OsSSI2 (OsSSI2-kd) reduced the oleic acid (18:1) level and increased that of stearic acid (18:0), indicating that OsSSI2 is responsible for fatty-acid desaturase activity. These plants displayed spontaneous lesion formation in leaf blades, retarded growth, slight increase in endogenous free salicylic acid (SA) levels, and SA/benzothiadiazole (BTH)-specific inducible genes, including WRKY45, a key regulator of SA/BTH-induced resistance, in rice. Moreover, the OsSSI2-kd plants showed markedly enhanced resistance to the blast fungus Magnaporthe grisea and leaf-blight bacteria Xanthomonas oryzae pv. oryzae. These results suggest that OsSSI2 is involved in the negative regulation of defense responses in rice, as are its Arabidopsis and soybean counterparts. Microarray analyses identified 406 genes that were differentially expressed (>or=2-fold) in OsSSI2-kd rice plants compared with wild-type rice and, of these, approximately 39% were BTH responsive. Taken together, our results suggest that induction of SA-responsive genes, including WRKY45, is likely responsible for enhanced disease resistance in OsSSI2-kd rice plants.

A Multi-year Beneficial Effect of Seed Priming with Gibberellic Acid-3 (GA3) on Plant Growth and Production in a Perennial Grass, Leymus chinensis.

Seed priming is a widely used technique in crops to obtain uniform germination and high-quality seedlings. In this study, we found a long-term effect of seed priming with gibberellic acid-3 (GA3) on plant growth and production in Leymus chinensis. Seeds were germinated on agar plates containing 0-200 µM GA3, and the germinated seedlings were transplanted to clay planting pots and grown for about one year. The clonal tillers grown from the mother plants were transplanted to field conditions in the second year. Results showed that GA3 treatment significantly increased seed germination rate by 14-27%. GA3 treatment also promoted subsequent plant growth and biomass production, as shown by a significant increase in plant height, tiller number, and fresh and dry weight in both pot (2016) and field (2017) conditions. It is particularly noteworthy that the growth-promoting effect of a single seed treatment with GA3 lasted for at least two years. In particular, GA3 treatment at 50 µM increased aboveground fresh and dry weight by 168.2% and 108.9% in pot-grown conditions, and 64.5% and 126.2% in field-grown conditions, respectively. These results imply a transgenerational transmission mechanism for the GA-priming effect on clonal offspring growth and biomass production in L. chinensis.

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.