OsCPK12 phosphorylates OsCATA and OsCATC to regulate H2O2 homeostasis and improve oxidative stress tolerance in rice.

Calcium-dependent protein kinases (CPKs), the best-characterized calcium sensors in plants, regulate many aspects of plant growth and development as well as plant adaptation to biotic and abiotic stresses. However, how CPKs regulate the antioxidant defense system remains largely unknown. We previously found that impaired function of OsCPK12 leads to oxidative stress in rice, with more H2O2, lower catalase (CAT) activity, and lower yield. Here, we explored the roles of OsCPK12 in oxidative stress tolerance in rice. Our results show that OsCPK12 interacts with and phosphorylates OsCATA and OsCATC at Ser11. Knockout of either OsCATA or OsCATC leads to an oxidative stress phenotype accompanied by higher accumulation of H2O2. Overexpression of the phosphomimetic proteins OsCATAS11D and OsCATCS11D in oscpk12-cr reduced the level of H2O2 accumulation. Moreover, OsCATAS11D and OsCATCS11D showed enhanced catalase activity in vivo and in vitro. OsCPK12-overexpressing plants exhibited higher CAT activity as well as higher tolerance to oxidative stress. Our findings demonstrate that OsCPK12 affects CAT enzyme activity by phosphorylating OsCATA and OsCATC at Ser11 to regulate H2O2 homeostasis, thereby mediating oxidative stress tolerance in rice.

Photoperiod and gravistimulation-associated Tiller Angle Control 1 modulates dynamic changes in rice plant architecture.

KEY MESSAGE: TAC1 is involved in photoperiodic and gravitropic responses to modulate rice dynamic plant architecture likely by affecting endogenous auxin distribution, which could explain TAC1 widespread distribution in indica rice. Plants experience a changing environment throughout their growth, which requires dynamic adjustments of plant architecture in response to these environmental cues. Our previous study demonstrated that Tiller Angle Control 1 (TAC1) modulates dynamic changes in plant architecture in rice; however, the underlying regulatory mechanisms remain largely unknown. In this study, we show that TAC1 regulates plant architecture in an expression dose-dependent manner, is highly expressed in stems, and exhibits dynamic expression in tiller bases during the growth period. Photoperiodic treatments revealed that TAC1 expression shows circadian rhythm and is more abundant during the dark period than during the light period and under short-day conditions than under long-day conditions. Therefore, it contributes to dynamic plant architecture under long-day conditions and loose plant architecture under short-day conditions. Gravity treatments showed that TAC1 is induced by gravistimulation and negatively regulates shoot gravitropism, likely by affecting auxin distribution. Notably, the tested indica rice containing TAC1 displayed dynamic plant architecture under natural long-day conditions, likely explaining the widespread distribution of TAC1 in indica rice. Our results provide new insights into TAC1-mediated regulatory mechanisms for dynamic changes in rice plant architecture.

Mutation of DEFECTIVE EMBRYO SAC1 results in a low seed-setting rate in rice by regulating embryo sac development.

The seed-setting rate has a significant effect on grain yield in rice (Oryza sativa L.). Embryo sac development is essential for seed setting; however, the molecular mechanism underlying this process remains unclear. Here, we isolated defective embryo sac1 (des1), a rice mutant with a low seed-setting rate. Cytological examination showed degenerated embryo sacs and reduced fertilization capacity in des1. Map-based cloning revealed a nonsense mutation in OsDES1, a gene that encodes a putative nuclear envelope membrane protein (NEMP)-domain-containing protein that is preferentially expressed in pistils. The OsDES1 mutation disrupts the normal formation of functional megaspores, which ultimately results in a degenerated embryo sac in des1. Reciprocal crosses showed that fertilization is abnormal and that the female reproductive organ is defective in des1. OsDES1 interacts with LONELY GUY (LOG), a cytokinin-activating enzyme that acts in the final step of cytokinin synthesis; mutation of LOG led to defective female reproductive organ development. These results demonstrate that OsDES1 functions in determining the rice seed-setting rate by regulating embryo sac development and fertilization. Our study sheds light on the function of NEMP-type proteins in rice reproductive development.

The clock component OsLUX regulates rice heading through recruiting OsELF3-1 and OsELF4s to repress Hd1 and Ghd7.

INTRODUCTION: Circadian clocks coordinate internal physiology and external environmental factors to regulate cereals flowering, which is critical for reproductive growth and optimal yield determination. OBJECTIVES: In this study, we aimed to confirm the role of OsLUX in flowering time regulation in rice. Further research illustrates how the OsELF4s-OsELF3-1-OsLUX complex directly regulates flowering-related genes to mediate rice heading. METHODS: We identified a circadian gene OsLUX by the MutMap method. The transcription levels of flowering-related genes were evaluated in WT and oslux mutants. OsLUX forms OsEC (OsELF4s-OsELF3-1-OsLUX) complex were supported by yeast two-hybrid, pull down, BiFC, and luciferase complementation assays (LCA). The EMSA, Chip-qPCR, luciferase luminescence images, and relative LUC activity assays were performed to examine the targeted regulation of flowering genes by the OsEC (OsELF4s-OsELF3-1-OsLUX) complex. RESULTS: The circadian gene OsLUX encodes an MYB family transcription factor that functions as a vital circadian clock regulator and controls rice heading. Defect in OsLUX causes an extremely late heading phenotype under natural long-day and short-day conditions, and the function was further confirmed through genetic complementation, overexpression, and CRISPR/Cas9 knockout. OsLUX forms the OsEC (OsELF4s-OsELF3-1-OsLUX) complex by recruiting OsELF3-1 and OsELF4s, which were required to regulate rice heading. OsELF3-1 contributes to the translocation of OsLUX to the nucleus, and a compromised flowering phenotype results upon mutation of any component of the OsEC complex. The OsEC complex directly represses Hd1 and Ghd7 expression via binding to their promoter's LBS (LUX binding site) element. CONCLUSION: Our findings show that the circadian gene OsLUX regulates rice heading by directly regulating rhythm oscillation and core flowering-time-related genes. We uncovered a mechanism by which the OsEC target suppresses the expression of Hd1 and Ghd7 directly to modulate photoperiodic flowering in rice. The OsEC (OsELF4s-OsELF3-1-OsLUX)-Hd1/Ghd7 regulatory module provides the genetic targets for crop improvement.

ERF Transcription Factor OsBIERF3 Positively Contributes to Immunity against Fungal and Bacterial Diseases but Negatively Regulates Cold Tolerance in Rice.

We previously showed that overexpression of the rice ERF transcription factor gene OsBIERF3 in tobacco increased resistance against different pathogens. Here, we report the function of OsBIERF3 in rice immunity and abiotic stress tolerance. Expression of OsBIERF3 was induced by Xanthomonas oryzae pv. oryzae, hormones (e.g., salicylic acid, methyl jasmonate, 1-aminocyclopropane-1-carboxylic acid, and abscisic acid), and abiotic stress (e.g., drought, salt and cold stress). OsBIERF3 has transcriptional activation activity that depends on its C-terminal region. The OsBIERF3-overexpressing (OsBIERF3-OE) plants exhibited increased resistance while OsBIERF3-suppressed (OsBIERF3-Ri) plants displayed decreased resistance to Magnaporthe oryzae and X. oryzae pv. oryzae. A set of genes including those for PRs and MAPK kinases were up-regulated in OsBIERF3-OE plants. Cell wall biosynthetic enzyme genes were up-regulated in OsBIERF3-OE plants but down-regulated in OsBIERF3-Ri plants; accordingly, cell walls became thicker in OsBIERF3-OE plants but thinner in OsBIERF3-Ri plants than WT plants. The OsBIERF3-OE plants attenuated while OsBIERF3-Ri plants enhanced cold tolerance, accompanied by altered expression of cold-responsive genes and proline accumulation. Exogenous abscisic acid and 1-aminocyclopropane-1-carboxylic acid, a precursor of ethylene biosynthesis, restored the attenuated cold tolerance in OsBIERF3-OE plants while exogenous AgNO3, an inhibitor of ethylene action, significantly suppressed the enhanced cold tolerance in OsBIERF3-Ri plants. These data demonstrate that OsBIERF3 positively contributes to immunity against M. oryzae and X. oryzae pv. oryzae but negatively regulates cold stress tolerance in rice.

The OsMPK15 Negatively Regulates Magnaporthe oryza and Xoo Disease Resistance via SA and JA Signaling Pathway in Rice.

Mitogen-activated protein kinase (MAPK) cascades play central roles in response to biotic and abiotic stresses. However, the mechanisms by which various MAPK members regulate the plant immune response in rice remain elusive. In this article, to characterize the mechanisms, the knock-out and overexpression mutants of OsMPK15 were constructed and the disease resistance was investigated under the various fungal and bacterial inoculations. The knock-out mutant of OsMPK15 resulted in the constitutive expression of pathogenesis-related (PR) genes, increased accumulation of reactive oxygen species (ROS) triggered by the pathogen-associated molecular pattern (PAMP) elicitor chitin, and significantly enhanced the disease resistance to different races of Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo), which cause the rice blast and bacterial blight diseases, respectively. On contrary, the expression of PR genes and ROS were down-regulated in the OsMPK15-overexpressing (OsMPK15-OE) lines. Meanwhile, phytohormones such as salicylic acid (SA) and jasmonic acid (JA) were accumulated in the mpk15 mutant lines but decreased in the OsMPK15-OE lines. The expression of SA- and JA-pathway associated genes were significantly upregulated in the mpk15 mutant, whereas it was down regulated in the OsMPK15-OE lines. We conclude that OsMPK15 may negatively regulate the disease resistance through modulating SA- and JA-mediated signaling pathway.

qSE7 is a major quantitative trait locus (QTL) influencing stigma exsertion rate in rice (Oryza sativa L.).

Stigma exsertion is a key determinant to increase the efficiency of commercial hybrid rice seed production. The major quantitative trait locus (QTL) qSE7 for stigma exsertion rate was previously detected on the chromosome 7 using 75 Chromosome Segment Substitution Lines (CSSLs) derived from a cross between the high stigma exsertion indica maintainer XieqingzaoB (XQZB) and low stigma exsertion indica restorer Zhonghui9308 (ZH9308). The C51 line, a CSSL population with an introgression from XQZB, was backcrossed with ZH9308 to produce the secondary F2 (BC5F2) and F2:3 (BC5F2:3) populations. As a result, the Near Isogenic Line (NIL qSE7XB) was developed. Analysis indicated qSE7 acted as a single Mendelian factor and decreased the stigma exsertion. We hypothesized qSE7 regulates single, dual, and total stigma exsertion rate, provided experimental support. qSE7 was mapped and localized between RM5436 and RM5499 markers, within a physical distance of 1000-kb. With use of new insertion-deletion (InDel) markers and analysis of the heterozygous and phenotypic data, it was ultimately dissected to a 322.9-kb region between InDel SER4-1 and RM5436. The results are useful for additional identification and isolation of this candidate gene controlling stigma exsertion rate, and provide a basis for further fine mapping, gene cloning, and Marker Assisted Selection (MAS) breeding later.

Premature leaf senescence 3, encoding a methyltransferase, is required for melatonin biosynthesis in rice.

Premature leaf senescence in rice is one of the most common factors affecting the plant's development and yield. Although methyltransferases are involved in diverse biological functions, their roles in rice leaf senescence have not been previously reported. In this study, we identified the premature leaf senescence 3 (pls3) mutant in rice, which led to early leaf senescence and early heading date. Further investigations revealed that premature leaf senescence was triggered by the accumulation of reactive oxygen species. Using physiological analysis, we found that chlorophyll content was reduced in the pls3 mutant leaves, while hydrogen peroxide (H2 O2 ) and malondialdehyde levels were elevated. Consistent with these findings, the pls3 mutant exhibited hypersensitivity to exogenous hydrogen peroxide. The expression of other senescence-associated genes such as Osh36 and RCCR1 was increased in the pls3 mutant. Positional cloning indicated the pls3 phenotype was the result of a mutation in OsMTS1, which encodes an O-methyltransferase in the melatonin biosynthetic pathway. Functional complementation of OsMTS1 in pls3 completely restored the wild-type phenotype. We found leaf melatonin content to be dramatically reduced in pls3, and that exogenous application of melatonin recovered the pls3 mutant's leaf senescence phenotype to levels comparable to that of wild-type rice. Moreover, overexpression of OsMTS1 in the wild-type plant increased the grain yield by 15.9%. Our results demonstrate that disruption of OsMTS1, which codes for a methyltransferase, can trigger leaf senescence as a result of decreased melatonin production.

Overexpression of MoSM1, encoding for an immunity-inducing protein from Magnaporthe oryzae, in rice confers broad-spectrum resistance against fungal and bacterial diseases.

Potential of MoSM1, encoding for a cerato-platanin protein from Magnaporthe oryzae, in improvement of rice disease resistance was examined. Transient expression of MoSM1 in rice leaves initiated hypersensitive response and upregulated expression of defense genes. When transiently expressed in tobacco leaves, MoSM1 targeted to plasma membrane. The MoSM1-overexpressing (MoSM1-OE) transgenic rice lines showed an improved resistance, as revealed by the reduced disease severity and decreased in planta pathogen growth, against 2 strains belonging to two different races of M. oryzae, causing blast disease, and against 2 strains of Xanthomonas oryzae pv. oryzae, causing bacterial leaf blight disease. However, no alteration in resistance to sheath blight disease was observed in MoSM1-OE lines. The MoSM1-OE plants contained elevated levels of salicylic acid (SA) and jasmonic acid (JA) and constitutively activated the expression of SA and JA signaling-related regulatory and defense genes. Furthermore, the MoSM1-OE plants had no effect on drought and salt stress tolerance and on grain yield. We conclude that MoSM1 confers a broad-spectrum resistance against different pathogens through modulating SA- and JA-mediated signaling pathways without any penalty on abiotic stress tolerance and grain yield, providing a promising potential for application of MoSM1 in improvement of disease resistance in crops.

Rice NAC transcription factor ONAC095 plays opposite roles in drought and cold stress tolerance.

BACKGROUND: The NAC (NAM, ATAF and CUC) transcriptional factors constitute a large family with more than 150 members in rice and some of them have been demonstrated to play crucial roles in plant abiotic stress response. Here, we report the characterization of a rice stress-responsive NAC gene, ONAC095, and the exploration of its function in drought and cold stress tolerance. RESULTS: Expression of ONAC095 was up-regulated by drought stress and abscisic acid (ABA) but down-regulated by cold stress. ONAC095 protein had transactivation activity and the C2 domain in C-terminal was found to be critical for transactivation activity. Transgenic rice lines with overexpression of ONAC095 (ONAC095-OE) and dominant chimeric repressor-mediated suppression of ONAC095 (ONAC095-SRDX) were generated. The ONAC095-OE plants showed comparable phenotype to wild type under drought and cold stress conditions. However, the ONAC095-SRDX plants displayed an improved drought tolerance but exhibited an attenuated cold tolerance. The ONAC095-SRDX plants had decreased water loss rate, increased proline and soluble sugar contents, and up-regulated expression of drought-responsive genes under drought condition, whereas the ONAC095-SRDX plants accumulated excess reactive oxygen species, increased malondialdehyde content and down-regulated expression of cold-responsive genes under cold condition. Furthermore, ONAC095-SRDX plants showed an increased ABA sensitivity, contained an elevated ABA level, and displayed altered expression of ABA biosynthetic and metabolic genes as well as some ABA signaling-related genes. CONCLUSION: Functional analyses through dominant chimeric repressor-mediated suppression of ONAC095 demonstrate that ONAC095 plays opposite roles in drought and cold stress tolerance, acting as a negative regulator of drought response but as a positive regulator of cold response in rice.

Virus-Induced Gene Silencing-Based Functional Analyses Revealed the Involvement of Several Putative Trehalose-6-Phosphate Synthase/Phosphatase Genes in Disease Resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 in Tomato.

Trehalose and its metabolism have been demonstrated to play important roles in control of plant growth, development, and stress responses. However, direct genetic evidence supporting the functions of trehalose and its metabolism in defense response against pathogens is lacking. In the present study, genome-wide characterization of putative trehalose-related genes identified 11 SlTPSs for trehalose-6-phosphate synthase, 8 SlTPPs for trehalose-6-phosphate phosphatase and one SlTRE1 for trehalase in tomato genome. Nine SlTPSs, 4 SlTPPs, and SlTRE1 were selected for functional analyses to explore their involvement in tomato disease resistance. Some selected SlTPSs, SlTPPs, and SlTRE1 responded with distinct expression induction patterns to Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 as well as to defense signaling hormones (e.g., salicylic acid, jasmonic acid, and a precursor of ethylene). Virus-induced gene silencing-mediated silencing of SlTPS3, SlTPS4, or SlTPS7 led to deregulation of ROS accumulation and attenuated the expression of defense-related genes upon pathogen infection and thus deteriorated the resistance against B. cinerea or Pst DC3000. By contrast, silencing of SlTPS5 or SlTPP2 led to an increased expression of the defense-related genes upon pathogen infection and conferred an increased resistance against Pst DC3000. Silencing of SlTPS3, SlTPS4, SlTPS5, SlTPS7, or SlTPP2 affected trehalose level in tomato plants with or without infection of B. cinerea or Pst DC3000. These results demonstrate that SlTPS3, SlTPS4, SlTPS5, SlTPS7, and SlTPP2 play roles in resistance against B. cinerea and Pst DC3000, implying the importance of trehalose and tis metabolism in regulation of defense response against pathogens in tomato.

BOTRYTIS-INDUCED KINASE1, a plasma membrane-localized receptor-like protein kinase, is a negative regulator of phosphate homeostasis in Arabidopsis thaliana.

BACKGROUND: Plants have evolved complex coordinated regulatory networks to cope with deficiency of phosphate (Pi) in their growth environment; however, the detailed molecular mechanisms that regulate Pi sensing and signaling pathways are not fully understood yet. We report here that the involvement of Arabidopsis BIK1, a plasma membrane-localized receptor-like protein kinase that plays critical role in immunity, in Pi starvation response. RESULTS: qRT-PCR analysis revealed that expression of BIK1 was induced by Pi starvation and GUS staining indicated that the BIK1 promoter activity was detected in root, stem and leaf tissues of plants grown in Pi starvation condition, demonstrating that BIK1 is responsive to Pi starvation stress. The bik1 plants accumulated higher Pi content in root and leaf tissues and exhibited altered root architecture such as shorter primary roots, longer and more root hairs and lateral roots, as compared with those in the wild type plants, when grown under Pi sufficient and deficient conditions. Increased anthocyanin content and acid phosphatase activity, reduced accumulation of reactive oxygen species and downregulated expression of Pi starvation-induced genes including PHR1, WRKY75, AT4, PHT1;2 and PHT1;4 were observed in bik1 plants grown under Pi deficient condition. Furthermore, the expression of PHO2 was downregulated while the expression of miRNA399a and miRNA399d, which target to PHO2, was upregulated in bik1 plants, compared to the wild type plants, when grown under Pi deficient condition. CONCLUSION: Our results demonstrate that BIK1 is a Pi starvation-responsive gene that functions as a negative regulator of Pi homeostasis in Arabidopsis.

Arabidopsis AtERF014 acts as a dual regulator that differentially modulates immunity against Pseudomonas syringae pv. tomato and Botrytis cinerea.

ERF transcription factors play critical roles in plant immune responses. Here, we report the function of AtERF014, a nucleus-localized transcriptional activator, in Arabidopsis immunity. Expression of AtERF014 was induced by Pseudomonas syringae pv. tomato (Pst) and Botrytis cinerea (Bc). AtERF014-overexpressing (OE) plants displayed increased Pst resistance but decreased Bc resistance, whereas AtERF014-RNAi plants exhibited decreased Pst resistance but increased Bc resistance. After Pst infection, expression of salicylic acid (SA)-responsive genes AtPR1 and AtPR5 in AtERF014-OE plants and of a jasmonic acid/ethylene-responsive gene AtPDF1.2 in AtERF014-RNAi plants was intensified but expression of AtPDF1.2 in AtERF014-OE plants and of AtPR1 and AtPR5 in AtERF014-RNAi plants was weakened. After Bc infection, expression of AtPR1 and AtPR5 in AtERF014-OE plants was attenuated but expression of AtPR1, AtPR5 and AtPDF1.2 in AtERF014-RNAi plants was strengthened. Pathogen- and flg22-induced ROS burst, expression of PTI genes and SA-induced defense were partially suppressed in AtERF014-RNAi plants, whereas pathogen-induced ROS and flg22-induced immune response were strengthened in AtER014-OE plants. Altered expression of AtERR014 affected expression of pectin biosynthetic genes and pectin content in AtERF014-RNAi plants was decreased. These data demonstrate that AtERF014 acts as a dual regulator that differentially modulates immunity against Pst and Bc in Arabidopsis.

Overexpression of a Stress-Responsive NAC Transcription Factor Gene ONAC022 Improves Drought and Salt Tolerance in Rice.

The NAC transcription factors play critical roles in regulating stress responses in plants. However, the functions for many of the NAC family members in rice are yet to be identified. In the present study, a novel stress-responsive rice NAC gene, ONAC022, was identified. Expression of ONAC022 was induced by drought, high salinity, and abscisic acid (ABA). The ONAC022 protein was found to bind specifically to a canonical NAC recognition cis-element sequence and showed transactivation activity at its C-terminus in yeast. The ONAC022 protein was localized to nucleus when transiently expressed in Nicotiana benthamiana. Three independent transgenic rice lines with overexpression of ONAC022 were generated and used to explore the function of ONAC022 in drought and salt stress tolerance. Under drought stress condition in greenhouse, soil-grown ONAC022-overexpressing (N22oe) transgenic rice plants showed an increased drought tolerance, leading to higher survival ratios and better growth than wild-type (WT) plants. When grown hydroponically in Hogland solution supplemented with 150 mM NaCl, the N22oe plants displayed an enhanced salt tolerance and accumulated less Na(+) in roots and shoots as compared to WT plants. Under drought stress condition, the N22oe plants exhibited decreased rates of water loss and transpiration, reduced percentage of open stomata and increased contents of proline and soluble sugars. However, the N22oe lines showed increased sensitivity to exogenous ABA at seed germination and seedling growth stages but contained higher level of endogenous ABA. Expression of some ABA biosynthetic genes (OsNCEDs and OsPSY), signaling and regulatory genes (OsPP2C02, OsPP2C49, OsPP2C68, OsbZIP23, OsAP37, OsDREB2a, and OsMYB2), and late stress-responsive genes (OsRAB21, OsLEA3, and OsP5CS1) was upregulated in N22oe plants. Our data demonstrate that ONAC022 functions as a stress-responsive NAC with transcriptional activator activity and plays a positive role in drought and salt stress tolerance through modulating an ABA-mediated pathway.

Tomato SlERF.A1, SlERF.B4, SlERF.C3 and SlERF.A3, Members of B3 Group of ERF Family, Are Required for Resistance to Botrytis cinerea.

The Ethylene-Responsive Factors (ERFs) comprise a large family of transcriptional factors that play critical roles in plant immunity. Gray mold disease caused by Botrytis cinerea, a typical necrotrophic fungal pathogen, is the serious disease that threatens tomato production worldwide. However, littler is known about the molecular mechanism regulating the immunity to B. cinerea in tomato. In the present study, virus-induced gene silencing (VIGS)-based functional analyses of 18 members of B3 group (also called Group IX) in tomato ERF family were performed to identify putative ERFs that are involved in disease resistance against B. cinerea. VIGS-based silencing of either SlERF.B1 or SlERF.C2 had lethal effect while silencing of SlERF.A3 (Pit4) significantly suppressed vegetative growth of tomato plants. Importantly, silencing of SlERF.A1, SlERF.A3, SlERF.B4, or SlERF.C3 resulted in increased susceptibility to B. cinerea, attenuated the B. cinerea-induced expression of jasmonic acid/ethylene-mediated signaling responsive defense genes and promoted the B. cinerea-induced H2O2 accumulation. However, silencing of SlERF.A3 also decreased the resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 but silencing of SlERF.A1, SlERF.B4 or SlERF.C3 did not affect the resistance to this bacterial pathogen. Expression of SlERF.A1, SlERF.A3, SlERF.B4, or SlERF.C3 was induced by B. cinerea and by defense signaling hormones such as salicylic acid, methyl jasmonate, and 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor). SlERF.A1, SlERF.B4, SlERF.C3, and SlERF.A3 proteins were found to localize in nucleus of cells and possess transactivation activity in yeasts. These data suggest that SlERF.A1, SlERF.B4, and SlERF.C3, three previously uncharacterized ERFs in B3 group, and SlERF.A3, a previously identified ERF with function in immunity to Pst DC3000, play important roles in resistance against B. cinerea in tomato.

Characterization, expression patterns and functional analysis of the MAPK and MAPKK genes in watermelon (Citrullus lanatus).

BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades, which consist of three functionally associated protein kinases, namely MEKKs, MKKs and MPKs, are universal signaling modules in all eukaryotes and have been shown to play critical roles in many physiological and biochemical processes in plants. However, little or nothing is known about the MPK and MKK families in watermelon. RESULTS: In the present study, we performed a systematic characterization of the ClMPK and ClMKK families including the identification and nomenclature, chromosomal localization, phylogenetic relationships, ClMPK-ClMKK interactions, expression patterns in different tissues and in response to abiotic and biotic stress and transient expression-based functional analysis for their roles in disease resistance. Genome-wide survey identified fifteen ClMPK and six ClMKK genes in watermelon genome and phylogenetic analysis revealed that both of the ClMPK and ClMKK families can be classified into four distinct groups. Yeast two-hybrid assays demonstrated significant interactions between members of the ClMPK and ClMKK families, defining putative ClMKK2-1/ClMKK6-ClMPK4-1/ClMPK4-2/ClMPK13 and ClMKK5-ClMPK6 cascades. Most of the members in the ClMPK and ClMKK families showed differential expression patterns in different tissues and in response to abiotic (e.g. drought, salt, cold and heat treatments) and biotic (e.g. infection of Fusarium oxysporum f. sp. niveum) stresses. Transient expression of ClMPK1, ClMPK4-2 and ClMPK7 in Nicotiana benthamiana resulted in enhanced resistance to Botrytis cinerea and upregulated expression of defense genes while transient expression of ClMPK6 and ClMKK2-2 led to increased susceptibility to B. cinerea. Furthermore, transient expression of ClMPK7 also led to hypersensitive response (HR)-like cell death and significant accumulation of H2O2 in N. benthamiana. CONCLUSION: We identified fifteen ClMPK and six ClMKK genes from watermelon and analyzed their phylogenetic relationships, expression patterns and protein-protein interactions and functions in disease resistance. Our results demonstrate that ClMPK1, ClMPK4-2 and ClMPK7 positively but ClMPK6 and ClMKK2-2 negatively regulate the resistance to B. cinerea when transiently expressed in N. benthamiana and that ClMPK7 functions as a regulator of HR-like cell death through modulating the generation of H2O2.

Tomato histone H2B monoubiquitination enzymes SlHUB1 and SlHUB2 contribute to disease resistance against Botrytis cinerea through modulating the balance between SA- and JA/ET-mediated signaling pathways.

BACKGROUND: Histone H2B monoubiquitination pathway has been shown to play critical roles in regulating growth/development and stress response in Arabidopsis. In the present study, we explored the involvement of the tomato histone H2B monoubiquitination pathway in defense response against Botrytis cinerea by functional analysis of SlHUB1 and SlHUB2, orthologues of the Arabidopsis AtHUB1/AtHUB2. METHODS: We used the TRV-based gene silencing system to knockdown the expression levels of SlHUB1 or SlHUB2 in tomato plants and compared the phenotype between the silenced and the control plants after infection with B. cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000. Biochemical and interaction properties of proteins were examined using in vitro histone monoubiquitination and yeast two-hybrid assays, respectively. The transcript levels of genes were analyzed by quantitative real time PCR (qRT-PCR). RESULTS: The tomato SlHUB1 and SlHUB2 had H2B monoubiquitination E3 ligases activity in vitro and expression of SlHUB1 and SlHUB2 was induced by infection of B. cinerea and Pst DC3000 and by treatment with salicylic acid (SA) and 1-amino cyclopropane-1-carboxylic acid (ACC). Silencing of either SlHUB1 or SlHUB2 in tomato plants showed increased susceptibility to B. cinerea, whereas silencing of SlHUB1 resulted in increased resistance against Pst DC3000. SlMED21, a Mediator complex subunit, interacted with SlHUB1 but silencing of SlMED21 did not affect the disease resistance to B. cinerea and Pst DC3000. The SlHUB1- and SlHUB2-silenced plants had thinner cell wall but increased accumulation of reactive oxygen species (ROS), increased callose deposition and exhibited altered expression of the genes involved in phenylpropanoid pathway and in ROS generation and scavenging system. Expression of genes in the SA-mediated signaling pathway was significantly upregulated, whereas expression of genes in the jasmonic acid (JA)/ethylene (ET)-mediated signaling pathway were markedly decreased in SlHUB1- and SlHUB2-silenced plants after infection of B. cinerea. CONCLUSION: VIGS-based functional analyses demonstrate that both SlHUB1 and SlHUB2 contribute to resistance against B. cinerea most likely through modulating the balance between the SA- and JA/ET-mediated signaling pathways.

Co-silencing of tomato S-adenosylhomocysteine hydrolase genes confers increased immunity against Pseudomonas syringae pv. tomato DC3000 and enhanced tolerance to drought stress.

S-adenosylhomocysteine hydrolase (SAHH), catalyzing the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and homocysteine, is a key enzyme that maintain the cellular methylation potential in all organisms. We report here the biological functions of tomato SlSAHHs in stress response. The tomato genome contains three SlSAHH genes that encode SlSAHH proteins with high level of sequence identity. qRT-PCR analysis revealed that SlSAHHs responded with distinct expression induction patterns to Pseudomonas syringae pv. tomato (Pst) DC3000 and Botrytis cinerea as well as to defense signaling hormones such as salicylic acid, jasmonic acid and a precursor of ethylene. Virus-induced gene silencing-based knockdown of individual SlSAHH gene did not affect the growth performance and the response to Pst DC3000. However, co-silencing of three SlSAHH genes using a conserved sequence led to significant inhibition of vegetable growth. The SlSAHH-co-silenced plants displayed increased resistance to Pst DC3000 but did not alter the resistance to B. cinerea. Co-silencing of SlSAHHs resulted in constitutively activated defense responses including elevated SA level, upregulated expression of defense-related and PAMP-triggered immunity marker genes and increased callose deposition and H2O2 accumulation. Furthermore, the SlSAHH-co-silenced plants also exhibited enhanced drought stress tolerance although they had relatively small roots. These data demonstrate that, in addition to the functions in growth and development, SAHHs also play important roles in regulating biotic and abiotic stress responses in plants.

Arabidopsis AtERF15 positively regulates immunity against Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea.

Upon pathogen infection, activation of immune response requires effective transcriptional reprogramming that regulates inducible expression of a large set of defense genes. A number of ethylene-responsive factor transcription factors have been shown to play critical roles in regulating immune responses in plants. In the present study, we explored the functions of Arabidopsis AtERF15 in immune responses against Pseudomonas syringae pv. tomato (Pst) DC3000, a (hemi)biotrophic bacterial pathogen, and Botrytis cinerea, a necrotrophic fungal pathogen. Expression of AtERF15 was induced by infection of Pst DC3000 and B. cinerea and by treatments with salicylic acid (SA) and methyl jasmonate. Biochemical assays demonstrated that AtERF15 is a nucleus-localized transcription activator. The AtERF15-overexpressing (AtERF15-OE) plants displayed enhanced resistance while the AtERF15-RNAi plants exhibited decreased resistance against Pst DC3000 and B. cinerea. Meanwhile, Pst DC3000- or B. cinerea-induced expression of defense genes was upregulated in AtERF15-OE plants but downregulated in AtERF15-RNAi plants, as compared to the expression in wild type plants. In response to infection with B. cinerea, the AtERF15-OE plants accumulated less reactive oxygen species (ROS) while the AtERF15-RNAi plants accumulated more ROS. The flg22- and chitin-induced oxidative burst was abolished and expression levels of the pattern-triggered immunity-responsive genes AtFRK1 and AtWRKY53 were suppressed in AtER15-RNAi plants upon treatment with flg22 or chitin. Furthermore, SA-induced defense response was also partially impaired in the AtERF15-RNAi plants. These data demonstrate that AtERF15 is a positive regulator of multiple layers of the immune responses in Arabidopsis.

Arabidopsis Raf-Like Mitogen-Activated Protein Kinase Kinase Kinase Gene Raf43 Is Required for Tolerance to Multiple Abiotic Stresses.

Mitogen-activated protein kinase (MAPK) cascades are critical signaling modules that mediate the transduction of extracellular stimuli into intracellular response. A relatively large number of MAPKKKs have been identified in a variety of plant genomes but only a few of them have been studied for their biological function. In the present study, we identified an Arabidopsis Raf-like MAPKKK gene Raf43 and studied its function in biotic and abiotic stress response using a T-DNA insertion mutant raf43-1 and two Raf43-overexpressing lines Raf43-OE#1 and Raf43-OE#13. Expression of Raf43 was induced by multiple abiotic and biotic stresses including treatments with drought, mannitol and oxidative stress or defense signaling molecule salicylic acid and infection with necrotrophic fungal pathogen Botrytis cinerea. Seed germination and seedling root growth of raf43-1 were significantly inhibited on MS medium containing mannitol, NaCl, H2O2 or methyl viologen (MV) while seed germination and seedling root growth of the Raf43-OE#1 and Raf43-OE#13 lines was similar to wild type Col-0 under the above stress conditions. Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress. Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines. Expression of stress-responsive RD17 and DREB2A genes was significantly down-regulated in raf43-1 plants. However, the raf43-1 and Raf43-overexpressing plants showed similar disease phenotype to the wild type plants after infection with B. cinerea or Pseudomonas syringae pv. tomato DC3000. Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis.