Fujian cytoplasmic male sterility and the fertility restorer gene OsRf19 provide a promising breeding system for hybrid rice.

Cytoplasmic male sterility (CMS) determined by mitochondrial genes and restorer of fertility (Rf) controlled by nuclear-encoded genes provide the breeding systems of many hybrid crops for the utilization of heterosis. Although several CMS/Rf systems have been widely exploited in rice, hybrid breeding using these systems has encountered difficulties due to either fertility instability or complications of two-locus inheritance or both. In this work, we characterized a type of CMS, Fujian Abortive cytoplasmic male sterility (CMS-FA), with stable sporophytic male sterility and a nuclear restorer gene that completely restores hybrid fertility. CMS is caused by the chimeric open reading frame FA182 that specifically occurs in the mitochondrial genome of CMS-FA rice. The restorer gene OsRf19 encodes a pentatricopeptide repeat (PPR) protein targeted to mitochondria, where it mediates the cleavage of FA182 transcripts, thus restoring male fertility. Comparative sequence analysis revealed that OsRf19 originated through a recent duplication in wild rice relatives, sharing a common ancestor with OsRf1a/OsRf5, a fertility restorer gene for Boro II and Hong-Lian CMS. We developed six restorer lines by introgressing OsRf19 into parental lines of elite CMS-WA hybrids; hybrids produced from these lines showed equivalent or better agronomic performance relative to their counterparts based on the CMS-WA system. These results demonstrate that CMS-FA/OsRf19 provides a highly promising system for future hybrid rice breeding.

From Green Super Rice to green agriculture: Reaping the promise of functional genomics research.

Producing sufficient food with finite resources to feed the growing global population while having a smaller impact on the environment has always been a great challenge. Here, we review the concept and practices of Green Super Rice (GSR) that have led to a paradigm shift in goals for crop genetic improvement and models of food production for promoting sustainable agriculture. The momentous achievements and global deliveries of GSR have been fueled by the integration of abundant genetic resources, functional gene discoveries, and innovative breeding techniques with precise gene and whole-genome selection and efficient agronomic management to promote resource-saving, environmentally friendly crop production systems. We also provide perspectives on new horizons in genomic breeding technologies geared toward delivering green and nutritious crop varieties to further enhance the development of green agriculture and better nourish the world population.

The Bnapus50K array: a quick and versatile genotyping tool for Brassica napus genomic breeding and research.

Rapeseed is a globally cultivated commercial crop, primarily grown for its oil. High-density single nucleotide polymorphism (SNP) arrays are widely used as a standard genotyping tool for rapeseed research, including for gene mapping, genome-wide association studies, germplasm resource analysis, and cluster analysis. Although considerable rapeseed genome sequencing data have been released, DNA arrays are still an attractive choice for providing additional genetic data in an era of high-throughput whole-genome sequencing. Here, we integrated re-sequencing DNA array data (32,216, 304 SNPs) from 505 inbred rapeseed lines, allowing us to develop a sensitive and efficient genotyping DNA array, Bnapus50K, with a more consistent genetic and physical distribution of probes. A total of 42,090 high-quality probes were filtered and synthesized, with an average distance between adjacent SNPs of 8 kb. To improve the practical application potential of this array in rapeseed breeding, we also added 1,618 functional probes related to important agronomic traits such as oil content, disease resistance, male sterility, and flowering time. The additional probes also included those specifically for detecting genetically modified material. These probes show a good detection efficiency and are therefore useful for gene mapping, along with crop variety improvement and identification. The novel Bnapus50K DNA array developed in this study could prove to be a quick and versatile genotyping tool for B. napus genomic breeding and research.

Genetic, transcriptional, and regulatory landscape of monolignol biosynthesis pathway in Miscanthus × giganteus.

BACKGROUND: Miscanthus × giganteus is widely recognized as a promising lignocellulosic biomass crop due to its advantages of high biomass production, low environmental impacts, and the potential to be cultivated on marginal land. However, the high costs of bioethanol production still limit the current commercialization of lignocellulosic bioethanol. The lignin in the cell wall and its by-products released in the pretreatment step is the main component inhibiting the enzymatic reactions in the saccharification and fermentation processes. Hence, genetic modification of the genes involved in lignin biosynthesis could be a feasible strategy to overcome this barrier by manipulating the lignin content and composition of M. × giganteus. For this purpose, the essential knowledge of these genes and understanding the underlying regulatory mechanisms in M. × giganteus is required. RESULTS: In this study, MgPAL1, MgPAL5, Mg4CL1, Mg4CL3, MgHCT1, MgHCT2, MgC3'H1, MgCCoAOMT1, MgCCoAOMT3, MgCCR1, MgCCR2, MgF5H, MgCOMT, and MgCAD were identified as the major monolignol biosynthetic genes in M. × giganteus based on genetic and transcriptional evidence. Among them, 12 genes were cloned and sequenced. By combining transcription factor binding site prediction and expression correlation analysis, MYB46, MYB61, MYB63, WRKY24, WRKY35, WRKY12, ERF021, ERF058, and ERF017 were inferred to regulate the expression of these genes directly. On the basis of these results, an integrated model was summarized to depict the monolignol biosynthesis pathway and the underlying regulatory mechanism in M. × giganteus. CONCLUSIONS: This study provides a list of potential gene targets for genetic improvement of lignocellulosic biomass quality of M. × giganteus, and reveals the genetic, transcriptional, and regulatory landscape of the monolignol biosynthesis pathway in M. × giganteus.

Transcriptome, physiological and biochemical analysis of Triarrhena sacchariflora in response to flooding stress.

BACKGROUND: In recent decades, the frequency of flooding is increasing with the change of global climate. Flooding has become one of the major abiotic stresses that seriously affect growth and development of plants. Triarrhena sacchariflora Nakai has been considered a promising energy crop for utilization in ethanol production. Flooding stress is among the most severe abiotic stressors in the production of Nakai. However, the physiological and molecular biological mechanisms of Nakai response to flooding is still unclear. In the present study, in order to understand the molecular mechanisms of Nakai in response to flooding stress, the transcriptome, physiological and biochemical were investigated. RESULTS: The results demonstrated that significant physiological changes were observed in photosynthetic system, antioxidative enzyme activity, chlorophyll, carotenoid, proline, lipid peroxidation and soluble sugar content under normal and flooding treatments. Such as, the chlorophyll, carotenoid contents and photosynthetic system were significantly decreased. Whereas, the antioxidative enzyme activity, proline, lipid peroxidation and soluble sugar has increased first and then decreased under treatments compared with the normal plants. Additionally, a total of 8832, 6608 and 3649 unigenes were validated to be differentially expressed under different treatments, respectively. Besides, gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of the different expression levels of genes also presented processes, which involved in photosynthesis, sucrose catabolism, glycolysis, stress response and defense, phytohormone biosynthesis and signal transduction. CONCLUSIONS: The results provide a comprehensive view of the complex molecular events involved in the response to flooding stress of Nakai leaves, which also will promote the research in the development of flood-resistant crops and provide new tools for Nakai breeders.

Selection of suitable reference genes for quantitive real-time PCR normalization in Miscanthus lutarioriparia.

Miscanthus lutarioriparia, which is found widespread in China, has attracted great attention as a most potential bioenergy plant for years. The quantitative real time PCR (RT-qPCR) has appeared as a sensitive and powerful technique to measure gene expression in living organisms during different development stages. In this study, we evaluated ten candidate genes, including 25S ribosomal RNA gene (25S rRNA), actin1 gene (ACT1), carotenoid-binding protein 20 gene (CBP20), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), Ubiquitin gene (UBQ), eukaryotic elongation factor 1-αgene (eEF-1α), α-tubulin gene (α-TUB), ß-tubulin gene (ß-TUB), eukaryotic translation initiation factor 4α-1 gene (eIF-4α) and NAC domain protein gene(NAC) in a series of 30 M. lutarioriparia samples followed by statistical algorithms geNorm and Normfinder to analyze the gene expression stability. The results indicated that eIF-4αand UBQ were the most stable expressed genes while CBP20 showed as the least stable among all the samples. Based on above research, we recommend that at least two top-ranked reference genes should be employed for expression data normalization. The best genes selected in this study will provide a starting point to select reference genes in the future in other tissues and under other experimental conditions in this energy crop candidate.

Transcriptomics and proteomics reveal genetic and biological basis of superior biomass crop Miscanthus.

Miscanthus is a rhizomatous C4 grass which is considered as potential high-yielding energy crop with the low-nutrient requirements, high water-use efficiency, and capability of C mitigation. To better understand the genetic basis, an integrative analysis of the transcriptome and proteome was performed to identify important genes and pathways involved in Miscanthus leaves. At the transcript level, 64,663 transcripts in M. lutarioriparius, 97,043 in M. sacchariflorus, 97,043 in M. sinensis, 67,323 in M. floridulus and 70,021 in M. × giganteus were detected by an RNA sequencing approach. At the protein level, 1964 peptide-represented proteins were identified and 1933 proteins differed by 1.5-fold or more in their relative abundance, as indicated by iTRAQ (isobaric tags for relative and absolute quantitation) analysis. Phylogenies were constructed from the nearly taxa of Miscanthus. A large number of genes closely related to biomass production were found. And SSR markers and their corresponding primers were derived from Miscanthus transcripts and 90% of them were successfully detected by PCR amplification among Miacanthus species. These similarities and variations on the transcriptional and proteomic level between Miscanthus species will serve as a resource for research in Miscanthus and other lignocellulose crops.

Nuclear DNA content in Miscanthus sp. and the geographical variation pattern in Miscanthus lutarioriparius.

The genome sizes of five Miscanthus species, including 79 accessions of M. lutarioriparius, 8 of M. floridulus, 6 of M. sacchariflorus, 7 of M. sinensis, and 4 of M. × giganteus were examined using flow cytometry. The overall average nuclear DNA content were 4.256 ± 0.6 pg/2C in M. lutarioriparius, 5.175 ± 0.3 pg/2C in M. floridulus, 3.956 ± 0.2 pg/2C in M. sacchariflorus, 5.272 ± 0.2 pg/2C in M. sinensis, and 6.932 ± 0.1 pg/2C in M. × giganteus. Interspecific variation was found at the diploid level, suggesting that DNA content might be a parameter that can be used to differentiate the species. Tetraploid populations were found in M. lutarioriparius, M. sacchariflorus, and M. sinensis, and their DNA content were 8.34 ± 1.2, 8.52, and 8.355 pg, respectively. The association between the DNA content of M. lutarioriparius, collected from representative ranges across the Yangtze River, and its geographic distribution was statistically analyzed. A consistent pattern of DNA content variation in 79 M. lutarioriparius accessions across its entire geographic range was found in this study. Along the Yangtze River, the DNA content of M. lutarioriparius tended to increase from the upstream to the downstream areas, and almost all tetraploids gathered in the upstream area extended to coastal regions.

Stacking S5-n and f5-n to overcome sterility in indica-japonica hybrid rice.

KEY MESSAGE: Pyramiding of S5 - n and f5 - n cumulatively improved seed-setting rate of indica-japonica hybrids, which provided an effective approach for utilization of inter-subspecific heterosis in rice breeding. Breeding for indica-japonica hybrid rice is an attractive approach to increase rice yield. However, hybrid sterility is a major obstacle in utilization of inter-subspecific heterosis. Wide-compatibility alleles can break the fertility barrier between indica and japonica subspecies, which have the potential to overcome inter-subspecific hybrid sterility. Here, we improved the compatibility of an elite indica restorer line 9311 to a broad spectrum of japonica varieties, by introducing two wide-compatibility alleles, S5-n and f5-n, regulating embryo-sac and pollen fertility, respectively. Through integrated backcross breeding, two near isogenic lines harboring either S5-n or f5-n and a pyramiding line carrying S5-n plus f5-n were obtained, with the recurrent parent genome recovery of 99.95, 99.49, and 99.44 %, respectively. The three lines showed normal fertility when crossed to typical indica testers. When testcrossed to five typical japonica varieties, these lines allowed significant increase of compatibility with constant agronomic performance. The introgressed S5-n could significantly improve 14.7-32.9 % embryo-sac fertility in indica-japonica hybrids. In addition, with the presence of f5-n fragment, S5-n would increase the spikelet fertility from 9.5 to 21.8 %. The introgressed f5-n fragment greatly improved anther dehiscence, embryo-sac and pollen fertility in indica-japonica hybrids, thus leading to improvement of spikelet fertility from 20.4 to 30.9 %. Moreover, the pyramiding line showed 33.6-46.7 % increase of spikelet fertility, suggesting cumulative effect of S5-n and f5-n fragment in seed-set improvement of inter-subspecific hybrids. Our results provided an effective approach for exploiting heterosis between indica and japonica subspecies, which had a profound implication in rice breeding.

Genome-wide Association Study (GWAS) of mesocotyl elongation based on re-sequencing approach in rice.

BACKGROUND: Mechanized dry seeded rice can save both labour and water resources. Rice seedling establishment is sensitive to sowing depth while mesocotyl elongation facilitates the emergence of deeply sown seeds. RESULTS: A set of 270 rice accessions, including 170 from the mini-core collection of Chinese rice germplasm (C Collection) and 100 varieties used in a breeding program for drought resistance (D Collection), was screened for mesocotyl lengths of seedlings grown in water (MLw) in darkness and in 5 cm sand culture (MLs). Twenty six accessions (10.53 %) have MLw longer than 1.0 cm. Eleven accessions had the highest mesocotyl lengths, i.e. 1.4 - 5.05 cm of MLw and 3.0 - 6.4 cm in 10 cm sand culture, including 7 upland landraces or varieties. The genotypic data of 1,019,883 SNPs were developed by re-sequencing of those accessions. A whole-genome SNP array (Rice SNP50) was used to genotype 24 accessions as a validation panel, giving 98.41 % of consistent SNPs with the re-sequencing data in average. GWAS based on compressed mixed linear model was conducted using GAPIT. Based on a threshold of -log(P) ≥8.0, 13 loci were associated to MLw on rice chromosome 1, 3, 4, 5, 6 and 9, respectively. Three associated loci, on chromosome 3, 6, and 10, were detected for MLs. A set of 99 associated SNPs for MLw, based on a compromised threshold (-log(P) ≥7.0), located in intergenic regions or different positions of 36 annotated genes, including one cullin and one growth regulating factor gene. CONCLUSIONS: Higher proportion and extension of elongated mesocotyls were observed in the mini-core collection of rice germplasm and upland rice landraces or varieties, possibly causing the correlation between mesocotyl elongation and drought resistance. GWAS found 13 loci for mesocotyl length measured in dark germination that confirmed the previously reported co-location of two QTLs across populations and experiments. Associated SNPs hit 36 annotated genes including function-matching candidates like cullin and GRF. The germplasm with elongated mesocotyl, especially upland landraces or varieties, and the associated SNPs could be useful in further studies and breeding of mechanized dry seeded rice.

Molecular cloning and characterization of two manganese superoxide dismutases from Miscanthus × giganteus.

KEY MESSAGE: Six MnSOD genes were isolated from five Miscanthus species. MgMnSOD1 functions in mitochondria and MgMnSOD1 seems to be the main MnSOD gene involved in stress response of M. × giganteus. Miscanthus × giganteus is a promising biomass energy crop with advantages of vigorous growth, high yield, low fertilizer and pesticide inputs. However, poor overwinter ability limits its widespread cultivation. Moreover, narrow genetic base may increase the risk of susceptibility to diseases and pests. Manganese superoxide dismutase (MnSOD), an important antioxidant enzyme involved in stress tolerance is able to protect plant cells from accumulated reactive oxygen species by converting superoxide to peroxide and oxygen. In many plants, overexpression of MnSOD has shown the ability to enhance the resistance to various stresses. This article describes the studies performed in an attempt to elucidate the molecular and enzymatic properties of MnSODs in M. × giganteus. MnSOD genes from M. × giganteus (MgMnSOD1, MgMnSOD2), M. lutarioriparia (MlMnSOD), M. sacchariflora (MsaMnSOD), M. sinensis (MsiMnSOD), and M. floridulus (MfMnSOD) were cloned and sequenced. The sequence analysis and expression patterns of MgMnSOD1 and MgMnSOD2 suggest that they were orthologous genes which were inherited from the two parents, M. sacchariflora and M. sinensis, respectively. In addition, MgMnSOD1 is predicted to be the main MnSOD gene involved in stress response of M. × giganteus. The activity of purified recombinant MgMnSOD1 was 1854.79 ± 39.98 U mg(-1) (mean ± SD). Further enzymatic assays revealed that the protein exhibited an outstanding thermal stability. MgMnSOD1 is predicted to be targeted to mitochondria and involved in removing the superoxide radical generated by respiration. The presence and sequences of other SOD isozymes transcripts were also investigated in this study.

Molecular breeding of thermo-sensitive genic male sterile (TGMS) lines of rice for blast resistance using Pi2 gene.

BACKGROUND: Blast disease caused by the fungal pathogen Magnaporthe oryzae is one of the big problems in rice production in China, especially for high yield hybrid varieties made from a two-line system in which thermo-sensitive genic male sterile (TGMS) lines are used. In this study, we report the introgression of a rice blast resistance gene Pi2 from VE6219 into C815S, an elite rice TGMS line, leading to the development of blast resistant TGMS lines through marker assisted selection (MAS) and phenotypic selection approaches. RESULTS: Four new TGMS lines with blast resistance gene Pi2 were developed from C815S (an elite TGMS line susceptible to the blast, used as recurrent parent) and VE6219 (a blast resistant line harboring Pi2, used as donor parent). The pathogenicity assays inoculated with 53 blast prevalent isolates in glasshouse showed that the blast resistant frequency of the four TGMS lines was 94.3%-98.1% that is equivalent to blast resistant donor parent VE6219. The field evaluation of the new lines and hybrids made from them at a blast epidemic site also showed high resistant levels against the blast. The genetic background of the newly developed TGMS lines were examined using a whole-genome single nucleotide polymorphism (SNP) array (RICE6K) that turned out more than 83% of the genomic markers were derived from the recurrent parent. The critical temperature points of fertility-sterility alteration of the new TGMS lines were between 22°C and 23°C of daily mean temperature, which is similar to that of C815S. The complete male sterility under natural growth conditions at Wuhan last more than 80 days. Their agronomic and grain quality traits meet the requirement for two-line hybrid rice production. CONCLUSIONS: The broad-spectrum and durable rice blast resistant gene Pi2 was introgressed into the elite TGMS line C815S background. The newly developed TGMS lines can be practically used for two-line hybrid rice breeding and must play an important role in sustainable rice production in China.

A high-density SNP genotyping array for rice biology and molecular breeding.

A high-density single nucleotide polymorphism (SNP) array is critically important for geneticists and molecular breeders. With the accumulation of huge amounts of genomic re-sequencing data and available technologies for accurate SNP detection, it is possible to design high-density and high-quality rice SNP arrays. Here we report the development of a high-density rice SNP array and its utility. SNP probes were designed by screening more than 10 000 000 SNP loci extracted from the re-sequencing data of 801 rice varieties and an array named RiceSNP50 was produced on the Illumina Infinium platform. The array contained 51 478 evenly distributed markers, 68% of which were within genic regions. Several hundred rice plants with parent/F1 relationships were used to generate a high-quality cluster file for accurate SNP calling. Application tests showed that this array had high genotyping accuracy, and could be used for different objectives. For example, a core collection of elite rice varieties was clustered with fine resolution. Genome-wide association studies (GWAS) analysis correctly identified a characterized QTL. Further, this array was successfully used for variety verification and trait introgression. As an accurate high-throughput genotyping tool, RiceSNP50 will play an important role in both functional genomics studies and molecular breeding.

A whole-genome SNP array (RICE6K) for genomic breeding in rice.

The advances in genotyping technology provide an opportunity to use genomic tools in crop breeding. As compared to field selections performed in conventional breeding programmes, genomics-based genotype screen can potentially reduce number of breeding cycles and more precisely integrate target genes for particular traits into an ideal genetic background. We developed a whole-genome single nucleotide polymorphism (SNP) array, RICE6K, based on Infinium technology, using representative SNPs selected from more than four million SNPs identified from resequencing data of more than 500 rice landraces. RICE6K contains 5102 SNP and insertion-deletion (InDel) markers, about 4500 of which were of high quality in the tested rice lines producing highly repeatable results. Forty-five functional markers that are located inside 28 characterized genes of important traits can be detected using RICE6K. The SNP markers are evenly distributed on the 12 chromosomes of rice with the average density of 12 SNPs per 1 Mb and can provide information for polymorphisms between indica and japonica subspecies as well as varieties within indica and japonica groups. Application tests of RICE6K showed that the array is suitable for rice germplasm fingerprinting, genotyping bulked segregating pools, seed authenticity check and genetic background selection. These results suggest that RICE6K provides an efficient and reliable genotyping tool for rice genomic breeding.

Development of genomics-based genotyping platforms and their applications in rice breeding.

Breeding by design has been an aspiration of researchers in the plant sciences for a decade. With the rapid development of genomics-based genotyping platforms and available of hundreds of functional genes/alleles in related to important traits, however, it may now be possible to turn this enduring ambition into a practical reality. Rice has a relatively simple genome comparing to other crops, and its genome composition and genetic behavior have been extensively investigated. Recently, rice has been taken as a model crop to perform breeding by design. The essential process of breeding by design is to integrate functional genes/alleles in an ideal genetic background, which requires high throughput genotyping platforms to screen for expected genotypes. With large amount of genome resequencing data and high-throughput genotyping technologies available, quite a number of genomics-based genotyping platforms have been developed. These platforms are widely used in genetic mapping, integration of target traits via marker-assisted backcrossing (MABC), pyramiding, recurrent selection (MARS) or genomic selection (GS). Here, we summarize and discuss recent exciting development of rice genomics-based genotyping platforms and their applications in molecular breeding.

Arabinose substitution degree in xylan positively affects lignocellulose enzymatic digestibility after various NaOH/H2SO4 pretreatments in Miscanthus.

Xylans are the major hemicelluloses in grasses, but their effects on biomass saccharification remain unclear. In this study, we examined the 79 representative Miscanthus accessions that displayed a diverse cell wall composition and varied biomass digestibility. Correlation analysis showed that hemicelluloses level has a strong positive effect on lignocellulose enzymatic digestion after NaOH or H(2)SO(4) pretreatment. Characterization of the monosaccharide compositions in the KOH-extractable and non-KOH-extractable hemicelluloses indicated that arabinose substitution degree of xylan is the key factor that positively affects biomass saccharification. The xylose/arabinose ratio after individual enzyme digestion revealed that the arabinose in xylan is partially associated with cellulose in the amorphous regions, which negatively affects cellulose crystallinity for high biomass digestibility. The results provide insights into the mechanism of lignocellulose enzymatic digestion upon pretreatment, and also suggest a goal for the genetic modification of hemicelluloses towards the bioenergy crop breeding of Miscanthus and grasses.

QTL scanning for rice yield using a whole genome SNP array.

High-throughput SNP genotyping is widely used for plant genetic studies. Recently, a RICE6K SNP array has been developed based on the Illumina Bead Array platform and Infinium SNP assay technology for genome-wide evaluation of allelic variations and breeding applications. In this study, the RICE6K SNP array was used to genotype a recombinant inbred line (RIL) population derived from the cross between the indica variety, Zhenshan 97, and the japonica variety, Xizang 2. A total of 3324 SNP markers of high quality were identified and were grouped into 1495 recombination bins in the RIL population. A high-density linkage map, consisting of the 1495 bins, was developed, covering 1591.2 cM and with average length of 1.1 cM per bin. Segregation distortions were observed in 24 regions of the 11 chromosomes in the RILs. One half of the distorted regions contained fertility genes that had been previously reported. A total of 23 QTLs were identified for yield. Seven QTLs were firstly detected in this study. The positive alleles from about half of the identified QTLs came from Zhenshan 97 and they had lower phenotypic values than Xizang 2. This indicated that favorable alleles for breeding were dispersed in both parents and pyramiding favorable alleles could develop elite lines. The size of the mapping population for QTL analysis using high throughput SNP genotyping platform is also discussed.

Identification of likely orthologs of tobacco salicylic acid-binding protein 2 and their role in systemic acquired resistance in Arabidopsis thaliana.

Salicylic acid-binding protein 2 (SABP2) is essential for the establishment of systemic acquired resistance (SAR) in tobacco; SABP2's methyl salicylate (MeSA) esterase activity is required in healthy systemic tissues of infected plants to release the active defense phytohormone SA from MeSA, which serves as a long-distance signal for SAR. In the current study, we characterize a new gene family from Arabidopsis thaliana encoding 18 potentially active alpha/beta fold hydrolases that share 32-57% identity with SABP2. Of 14 recombinant AtMES (MES for methyl esterase) proteins tested, five showed preference for MeSA as a substrate and displayed SA inhibition of MeSA esterase activity in vitro (AtMES1, -2, -4, -7, and -9). The two genes encoding MeSA esterases with the greatest activity, AtMES1 and -9, as well as AtMES7 were transcriptionally upregulated during infection of Arabidopsis with avirulent Pseudomonas syringae. In addition, conditional expression of AtMES1, -7, or -9 complemented SAR deficiency in SABP2-silenced tobacco, suggesting that these three members of the AtMES family are SABP2 functional homologs (orthologs). Underexpression by knockout mutation and/or RNAi-mediated silencing of multiple AtMES genes, including AtMES1, -2, -7, and -9, compromised SAR in Arabidopsis and correlated with enhanced accumulation of MeSA in the systemic tissue of SAR-induced plants. Together, the data show that several members of the AtMES gene family are functionally homologous to SABP2 and redundant for MeSA hydrolysis and probably SAR. These data suggest that MeSA is a conserved SAR signal in Arabidopsis and tobacco.

The Arabidopsis gain-of-function mutant ssi4 requires RAR1 and SGT1b differentially for defense activation and morphological alterations.

A gain-of-function mutation in resistance (R) gene SSI4 causes constitutive activation of defense responses, spontaneous necrotic lesion formation, enhanced resistance against virulent pathogens, and a severe dwarf phenotype. Genetic analysis revealed that ssi4-induced H(2)O(2) accumulation and spontaneous cell death require RAR1, whereas ssi4-mediated stunting is dependent on SGT1b. By contrast, both RAR1 and SGT1b are required in a genetically additive manner for ssi4-induced disease resistance, SA accumulation, and lesion formation after pathogen infection. These data point to cooperative yet distinct functions of RAR1 and SGT1b in responses conditioned by a deregulated nucleotide-binding leucine-rich repeat protein. We also found that RAR1 and SGT1b together contribute to basal resistance because an ssi4 rar1 sgt1b triple mutant exhibited enhanced susceptibility to virulent pathogen infection compared with wild-type SSI4 plants. All ssi4-induced phenotypes were suppressed when plants were grown at 22 degrees C under high relative humidity. However, low temperature (16 degrees C) triggered ssi4-mediated cell death via an RAR1-dependent pathway even in the presence of high humidity. Thus, multiple environmental factors impact on ssi4 signaling, as has been observed for other constitutive defense mutants and R gene-triggered pathways.

High humidity suppresses ssi4-mediated cell death and disease resistance upstream of MAP kinase activation, H2O2 production and defense gene expression.

The Arabidopsis ssi4 mutant, which exhibits spontaneous lesion formation, constitutive expression of pathogenesis-related (PR) genes and enhanced resistance to virulent bacterial and oomycete pathogens, contains a gain-of-function mutation in a TIR-NBS-LRR type R gene. Epistatic analyses revealed that both PR gene expression and disease resistance are activated via a salicylic acid (SA)- and EDS1-dependent, but NPR1- and NDR1-independent signaling pathway. In this study, we demonstrate that in moderate relative humidity (RH; 60%), the ssi4 mutant accumulates H(2)O(2) and SA prior to lesion formation and displays constitutive activation of the MAP kinases AtMPK6 and AtMPK3. It also constitutively expresses a variety of defense-associated genes, including those encoding the WRKY transcription factors AtWRKY29 and AtWRKY6, the MAP kinases AtMPK6 and AtMPK3, the powdery mildew R proteins RPW8.1 and RPW8.2, EDS1 and PR proteins. All of these ssi4-induced responses, as well as the chlorotic, stunted morphology and enhanced disease resistance phenotype, are suppressed by high RH (95%) growth conditions. Thus, a humidity sensitive factor (HSF) appears to function at an early point in the ssi4 signaling pathway. All ssi4 phenotypes, except for MAP kinase activation, also were suppressed by the eds1-1 mutation. Thus, ssi4-induced MAP kinase activation occurs downstream of the HSF but either upstream of EDS1 or on a separate branch of the ssi4 signaling pathway. SA is a critical signaling component in ssi4-mediated defense responses. However, exogenously supplied SA failed to restore lesion formation in high RH-grown ssi4 plants, although it induced defense gene expression. Thus, additional signals also are involved.