Fe2+ Ions Alleviate the Symptom of Citrus Greening Disease.

Citrus greening (CG) is among the most devastating citrus diseases worldwide. CG-infected trees exhibit interveinal chlorotic leaves due to iron (Fe) deficiency derived from CG; thus, Fe content is lower in infected leaves than in healthy leaves. In this study, we demonstrated that the foliar application of Fe2+ relieves the symptom of CG infection in citrus trees. We applied Fe2+ and citrate to the leaves of infected rough lemon plants. Following this treatment, a reduction in the number of yellow symptomatic leaves was observed, and their growth was restored. Using chlorophyll content as an index, we screened for effective Fe complexes and found that a high ratio of citrate to Fe2+ in the applied solution led to effects against CG in Shikuwasa trees. A high proportion of Fe2+ to total Fe was another key factor explaining the effectiveness of the solution in CG infection, indicating the importance of Fe2+ absorption into plant cells. We confirmed the proportion of Fe2+ to total Fe through the high correlation of reflectometry data via a triazine reaction and X-ray absorption fine structure analysis. These results demonstrate that the foliar application of a high-Fe2+ citrate solution can restore the growth of CG diseased trees.

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.

Panicle blast 1 (Pb1) resistance is dependent on at least four QTLs in the rice genome.

BACKGROUND: Rice blast is the most serious disease afflicting rice and there is an urgent need for the use of disease resistance (R) genes in blast tolerance breeding programs. Pb1 is classified as a quantitative resistance gene and it does not have fungal specificity. Pb1-mediated resistance develops in the latter stages of growth. However, some cultivars, such as Kanto209 (K209), cultivar name Satojiman, despite possessing Pb1, do not exert resistance to rice blast during the reproductive stage. RESULTS: We found that the expression of WRKY45 gene downstream of Pb1 was weakly induced by rice blast inoculation at the full heading stage in K209. Genetic analysis using the SNP-based Golden Gate assay of K209 crossing with Koshihikari Aichi SBL (KASBL) found at least four regions related to the resistance in the rice genome (Chr8, Chr9, Chr7, Chr11). Mapping of QTL related to Chr7 confirmed the existence of factors that were required for the resistance of Pb1 in the 22 to 23 Mbp region of the rice genome. CONCLUSION: We clarified how the K209 cultivar is vulnerable to the blast disease despite possessing Pb1 and found the DNA marker responsible for the quantitative resistance of Pb1. We identified the QTL loci required for Pb1-mediated resistance to rice panicle blast. Pb1 was negatively dependent on at least three QTLs, 7, 9 and 11, and positively dependent on one, QTL 8, in the K209 genome. This finding paves the way for creating a line to select optimal QTLs in order to make use of Pb1-mediated resistance more effectively.

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.

Transcription Factor WRKY62 Plays a Role in Pathogen Defense and Hypoxia-Responsive Gene Expression in Rice.

WRKY62 is a transcriptional repressor regulated downstream of WRKY45, a central transcription factor of the salicylic acid signaling pathway in rice. Previously, WRKY62 was reported to regulate defense negatively. However, our expressional analysis using WRKY62-knockdown rice indicated that WRKY62 positively regulates defense genes, including diterpenoid phytoalexin biosynthetic genes and their transcriptional regulator DPF. Blast and leaf blight resistance tests also showed that WRKY62 is a positive defense regulator. Yeast two-hybrid, co-immunoprecipitation and gel-shift assays showed that WRKY45 and WRKY62 can form a heterodimer, as well as homodimers, that bind to W-boxes in the DPF promoter. In transient assays in rice sheaths, the simultaneous introduction of WRKY45 and WRKY62 as effectors resulted in a strong activation of the DPF promoter:hrLUC reporter gene, whereas the activity declined with excessive WRKY62. Thus, the WRKY45-WRKY62 heterodimer acts as a strong activator, while the WRKY62 homodimer acts as a repressor. While benzothiadiazole induced equivalent numbers of WRKY45 and WRKY62 transcripts, consistent with heterodimer formation and DPF activation, submergence and nitrogen replacement induced only WRKY62 transcripts, consistent with WRKY62 homodimer formation and DPF repression. Moreover, WRKY62 positively regulated hypoxia genes, implying a role forWRKY62 in the modulation of the 'trade-off' between defense and hypoxia responses.

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.

Development of disease-resistant rice by optimized expression of WRKY45.

The rice transcription factor WRKY45 plays a central role in the salicylic acid signalling pathway and mediates chemical-induced resistance to multiple pathogens, including Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae. Previously, we reported that rice transformants overexpressing WRKY45 driven by the maize ubiquitin promoter were strongly resistant to both pathogens; however, their growth and yield were negatively affected because of the trade-off between the two conflicting traits. Also, some unknown environmental factor(s) exacerbated this problem. Here, we report the development of transgenic rice lines resistant to both pathogens and with agronomic traits almost comparable to those of wild-type rice. This was achieved by optimizing the promoter driving WRKY45 expression. We isolated 16 constitutive promoters from rice genomic DNA and tested their ability to drive WRKY45 expression. Comparisons among different transformant lines showed that, overall, the strength of WRKY45 expression was positively correlated with disease resistance and negatively correlated with agronomic traits. We conducted field trials to evaluate the growth of transgenic and control lines. The agronomic traits of two lines expressing WRKY45 driven by the OsUbi7 promoter (PO sUbi7 lines) were nearly comparable to those of untransformed rice, and both lines were pathogen resistant. Interestingly, excessive WRKY45 expression rendered rice plants sensitive to low temperature and salinity, and stress sensitivity was correlated with the induction of defence genes by these stresses. These negative effects were barely observed in the PO sUbi7 lines. Moreover, their patterns of defence gene expression were similar to those in plants primed by chemical defence inducers.

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.

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.

WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance.

OsWRKY76 encodes a group IIa WRKY transcription factor of rice. The expression of OsWRKY76 was induced within 48h after inoculation with rice blast fungus (Magnaporthe oryzae), and by wounding, low temperature, benzothiadiazole, and abscisic acid. Green fluorescent protein-fused OsWRKY76 localized to the nuclei in rice epidermal cells. OsWRKY76 showed sequence-specific DNA binding to the W-box element in vitro and exhibited W-box-mediated transcriptional repressor activity in cultured rice cells. Overexpression of OsWRKY76 in rice plants resulted in drastically increased susceptibility to M. oryzae, but improved tolerance to cold stress. Microarray analysis revealed that overexpression of OsWRKY76 suppresses the induction of a specific set of PR genes and of genes involved in phytoalexin synthesis after inoculation with blast fungus, consistent with the observation that the levels of phytoalexins in the transgenic rice plants remained significantly lower than those in non-transformed control plants. Furthermore, overexpression of OsWRKY76 led to the increased expression of abiotic stress-associated genes such as peroxidase and lipid metabolism genes. These results strongly suggest that OsWRKY76 plays dual and opposing roles in blast disease resistance and cold tolerance.

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.

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.

Nuclear ubiquitin proteasome degradation affects WRKY45 function in the rice defense program.

The transcriptional activator WRKY45 plays a major role in the salicylic acid/benzothiadiazole-induced defense program in rice. Here, we show that the nuclear ubiquitin-proteasome system (UPS) plays a role in regulating the function of WRKY45. Proteasome inhibitors induced accumulation of polyubiquitinated WRKY45 and transient up-regulation of WRKY45 target genes in rice cells, suggesting that WRKY45 is constantly degraded by the UPS to suppress defense responses in the absence of defense signals. Mutational analysis of the nuclear localization signal indicated that UPS-dependent WRKY45 degradation occurs in the nuclei. Interestingly, the transcriptional activity of WRKY45 after salicylic acid treatment was impaired by proteasome inhibition. The same C-terminal region in WRKY45 was essential for both transcriptional activity and UPS-dependent degradation. These results suggest that UPS regulation also plays a role in the transcriptional activity of WRKY45. It has been reported that AtNPR1, the central regulator of the salicylic acid pathway in Arabidopsis, is regulated by the UPS. We found that OsNPR1/NH1, the rice counterpart of NPR1, was not stabilized by proteasome inhibition under uninfected conditions. We discuss the differences in post-translational regulation of salicylic acid pathway components between rice and Arabidopsis.

Rice WRKY45 plays important roles in fungal and bacterial disease resistance.

Plant 'activators', such as benzothiadiazole (BTH), protect plants from various diseases by priming the plant salicylic acid (SA) signalling pathway. We have reported previously that a transcription factor identified in rice, WRKY45 (OsWRKY45), plays a pivotal role in BTH-induced disease resistance by mediating SA signalling. Here, we report further functional characterization of WRKY45. Different plant activators vary in their action points, either downstream (BTH and tiadinil) or upstream (probenazole) of SA. Rice resistance to Magnaporthe grisea, induced by both types of plant activator, was markedly reduced in WRKY45-knockdown (WRKY45-kd) rice, indicating a universal role for WRKY45 in chemical-induced resistance. Fungal invasion into rice cells was blocked at most attempted invasion sites (pre-invasive defence) in WRKY45-overexpressing (WRKY45-ox) rice. Hydrogen peroxide accumulated within the cell wall underneath invading fungus appressoria or between the cell wall and the cytoplasm, implying a possible role for H(2)O(2) in pre-invasive defence. Moreover, a hypersensitive reaction-like reaction was observed in rice cells, in which fungal growth was inhibited after invasion (post-invasive defence). The two levels of defence mechanism appear to correspond to Type I and II nonhost resistances. The leaf blast resistance of WRKY45-ox rice plants was much higher than that of other known blast-resistant varieties. WRKY45-ox plants also showed strong panicle blast resistance. BTH-induced resistance to Xanthomonas oryzae pv. oryzae was compromised in WRKY45-kd rice, whereas WRKY45-ox plants were highly resistant to this pathogen. However, WRKY45-ox plants were susceptible to Rhizoctonia solani. These results indicate the versatility and limitations of the application of this gene.

Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice.

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.

RiceFOX: a database of Arabidopsis mutant lines overexpressing rice full-length cDNA that contains a wide range of trait information to facilitate analysis of gene function.

Identification of gene function is important not only for basic research but also for applied science, especially with regard to improvements in crop production. For rapid and efficient elucidation of useful traits, we developed a system named FOX hunting (Full-length cDNA Over-eXpressor gene hunting) using full-length cDNAs (fl-cDNAs). A heterologous expression approach provides a solution for the high-throughput characterization of gene functions in agricultural plant species. Since fl-cDNAs contain all the information of functional mRNAs and proteins, we introduced rice fl-cDNAs into Arabidopsis plants for systematic gain-of-function mutation. We generated >30,000 independent Arabidopsis transgenic lines expressing rice fl-cDNAs (rice FOX Arabidopsis mutant lines). These rice FOX Arabidopsis lines were screened systematically for various criteria such as morphology, photosynthesis, UV resistance, element composition, plant hormone profile, metabolite profile/fingerprinting, bacterial resistance, and heat and salt tolerance. The information obtained from these screenings was compiled into a database named 'RiceFOX'. This database contains around 18,000 records of rice FOX Arabidopsis lines and allows users to search against all the observed results, ranging from morphological to invisible traits. The number of searchable items is approximately 100; moreover, the rice FOX Arabidopsis lines can be searched by rice and Arabidopsis gene/protein identifiers, sequence similarity to the introduced rice fl-cDNA and traits. The RiceFOX database is available at http://ricefox.psc.riken.jp/.

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.

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.

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.

Systematic approaches to using the FOX hunting system to identify useful rice genes.

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23,000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis. This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.