Yafen Zhang

Tomato WRKY transcriptional factor SlDRW1 is required for disease resistance against Botrytis cinerea and tolerance to oxidative stress

WRKY proteins comprise a large family of transcription factors that play important roles in plant responses to biotic and abiotic stresses; however, only a few of tomato WRKYs have been studied for their biological functions. In the present study, we identified a Botrytis cinerea-responsive WRKY gene SlDRW1 (Solanum lycopersicumdefense-related WRKY1) from tomato. SlDRW1 is a nucleus localized protein with transactivation activity in yeast. Expression of SlDRW1 was significantly induced by B. cinerea, leading to 10-13 folds of increase than that in the mock-inoculated plants but not by Pseudomonas syringae pv. tomato (Pst) DC3000. Silencing of SlDRW1 resulted in increased severity of disease caused by B. cinerea, but did not affect the phenotype of disease caused by Pst DC3000. In addition, silencing of SlDRW1 also resulted in decreased tolerance against oxidative stress but did not affect drought stress tolerance. Furthermore, silencing of SlDRW1 attenuated defense response such as expression of defense-related genes after infection by B. cinerea. Our results demonstrate that SlDRW1 is a positive regulator of defense response in tomato against B. cinerea and oxidative stress.

Tomato NAC Transcription Factor SlSRN1 Positively Regulates Defense Response against Biotic Stress but Negatively Regulates Abiotic Stress Response

Biotic and abiotic stresses are major unfavorable factors that affect crop productivity worldwide. NAC proteins comprise a large family of transcription factors that play important roles in plant growth and development as well as in responses to biotic and abiotic stresses. In a virus-induced gene silencing-based screening to identify genes that are involved in defense response against Botrytis cinerea, we identified a tomato NAC gene SlSRN1 (Solanum lycopersicum Stress-related NAC1). SlSRN1 is a plasma membrane-localized protein with transactivation activity in yeast. Expression of SlSRN1 was significantly induced by infection with B. cinerea or Pseudomonas syringae pv. tomato (Pst) DC3000, leading to 6–8 folds higher than that in the mock-inoculated plants. Expression of SlSRN1 was also induced by salicylic acid, jasmonic acid and 1-amino cyclopropane-1-carboxylic acid and by drought stress. Silencing of SlSRN1 resulted in increased severity of diseases caused by B. cinerea and Pst DC3000. However, silencing of SlSRN1 resulted in increased tolerance against oxidative and drought stresses. Furthermore, silencing of SlSRN1 accelerated accumulation of reactive oxygen species but attenuated expression of defense genes after infection by B. cinerea. Our results demonstrate that SlSRN1 is a positive regulator of defense response against B. cinerea and Pst DC3000 but is a negative regulator for oxidative and drought stress response in tomato.

Tomato SR/CAMTA transcription factors SlSR1 and SlSR3L negatively regulate disease resistance response and SlSR1L positively modulates drought stress tolerance

BackgroundThe SR/CAMTA proteins represent a small family of transcription activators that play important roles in plant responses to biotic and abiotic stresses. Seven SlSR/CAMTA genes were identified in tomato as tomato counterparts of SR/CAMTA; however, the involvement of SlSRs/CAMTAs in biotic and abiotic stress responses is not clear. In this study, we performed functional analysis of the SlSR/CAMTA family for their possible functions in defense response against pathogens and tolerance to drought stress.ResultsExpression of SlSRs was induced with distinct patterns by Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000. Virus-induced gene silencing (VIGS)-based knockdown of either SlSR1 or SlSR3L in tomato resulted in enhanced resistance to B. cinerea and Pst DC3000 and led to constitutive accumulation of H2O2, elevated expression of defense genes, marker genes for pathogen-associated molecular pattern-triggered immunity, and regulatory genes involved in the salicylic acid- and ethylene-mediated signaling pathways. Furthermore, the expression of SlSR1L and SlSR2L in detached leaves and whole plants was significantly induced by drought stress. Silencing of SlSR1L led to decreased drought stress tolerance, accelerated water loss in leaves, reduced root biomass and attenuated expression of drought stress responsive genes in tomato. The SlSR1 and SlSR3L proteins were localized in the nucleus of plant cells when transiently expressed in Nicotiana benthamiana and had transcriptional activation activity in yeast.ConclusionsVIGS-based functional analyses demonstrate that both SlSR1 and SlSR3L in the tomato SlSR/CAMTA family are negative regulators of defense response against B. cinerea and Pst DC3000 while SlSR1L is a positive regulator of drought stress tolerance in tomato.

Tomato SlMKK2 and SlMKK4 contribute to disease resistance against Botrytis cinerea

BackgroundMitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules that mediate the transduction of extracellular stimuli via receptors/sensors into intracellular responses and play key roles in plant immunity against pathogen attack. However, the function of tomato MAPK kinases, SlMKKs, in resistance against Botrytis cinerea remains unclear yet.ResultsA total of five SlMKK genes with one new member, SlMKK5, were identified in tomato. qRT-PCR analyses revealed that expression of SlMKK2 and SlMKK4 was strongly induced by B. cinerea and by jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Virus-induced gene silencing (VIGS)-based knockdown of individual SlMKKs and disease assays identified that SlMKK2 and SlMKK4 but not other three SlMKKs (SlMKK1, SlMKK3 and SlMKK5) are involved in resistance against B. cinerea. Silencing of SlMKK2 or SlMKK4 resulted in reduced resistance to B. cinerea, increased accumulation of reactive oxygen species and attenuated expression of defense genes after infection of B. cinerea in tomato plants. Furthermore, transient expression of constitutively active phosphomimicking forms SlMKK2DD and SlMKK4DD in leaves of Nicotiana benthamiana plants led to enhanced resistance to B. cinerea and elevated expression of defense genes.ConclusionsVIGS-based knockdown of SlMKK2 and SlMKK4 expression in tomato and gain-of-function transient expression of constitutively active phosphomimicking forms SlMKK2DD and SlMKK2DD in N. benthamiana demonstrate that both SlMKK2 and SlMKK4 function as positive regulators of defense response against B. cinerea.

Genome-Wide Identification, Biochemical Characterization, and Expression Analyses of the YTH Domain-Containing RNA-Binding Protein Family in Arabidopsis and Rice

RNA-binding proteins are critical to RNA metabolism in cells and, thus, play important roles in diverse biological processes. In the present study, we identified the YTH domain-containing RNA-binding protein (RBP) family in Arabidopsis thaliana and rice at the molecular and biochemical levels. A total of 13 and 12 genes were found to encode YTH domain-containing RBPs in Arabidopsis and rice and named as AtYTH01–13 and OsYTH01–12, respectively. The phylogeny, chromosomal location, and structures of genes and proteins were analyzed. Electrophoretic mobility shift assays demonstrated that recombinant AtYTH05 protein could bind to single-stranded RNA in vitro, demonstrating that the YTH proteins have RNA-binding activity. Analyses of publicly available microarray data, gene expression by qRT-PCR, and AtYTH05 promoter activity indicate that the Arabidopsis AtYTHs and rice OsYTHs genes have distinct and diverse expression patterns in different tissues and developmental stages, showing tissue- and developmental-specific expression patterns. Furthermore, analyses of publicly available microarray data also indicate that many of the Arabidopsis AtYTHs and rice OsYTHs genes might be involved in responses to various abiotic and biotic stresses as well as in response to hormones. Our data demonstrate that the YTH family proteins are a novel group of RBPs and provide useful clues to define their biological functions of this RBP family in plants.

Vitamin B6 contributes to disease resistance against Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea in Arabidopsis thaliana

Vitamin B6 (VB6) is an important cofactor for numerous enzymatic reactions and plays an important role in abiotic stress tolerance. However, direct molecular evidence supporting a role for VB6 in plant disease resistance remains lacking. In this study, we explored the possible function of VB6 in disease resistance by analyzing disease phenotypes of Arabidopsis mutants with defects in de novo biosynthetic pathway and salvage pathway of VB6 biosynthesis against Pseudomonas syringae pv. tomato (Pst) DC3000 and Botrytis cinerea. Mutations in AtPDX1.2 and AtPDX1.3 genes involved in the de novo pathway, and in AtSOS4 gene involved in the salvage pathway led to increased levels of diseases caused by Pst DC3000 and B. cinerea. The pdx1.2 and pdx1.3 plants had reduced VB6 contents and showed a further reduction in VB6 contents after infection by Pst DC3000 or B. cinerea. Our preliminary results suggest an important role for VB6 in plant disease resistance against different types of pathogens.

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.

BackgroundHistone 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.MethodsWe 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).ResultsThe 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.ConclusionVIGS-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.

Tomato SlMPK4 is required for resistance against Botrytis cinerea and tolerance to drought stress

Mitogen-activated protein kinases (MPKs) play important roles in biotic and abiotic stress responses. In the present study, we identified a tomato MPK gene, SlMPK4, a possible homolog of Arabidopsis AtMPK4, and performed functional analysis to examine its possible roles in biotic and abiotic responses. Expression of SlMPK4 was induced by infection with Botrytis cinerea and by exogenous application of jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Knockdown of the endogenous SlMPK4 expression through virus-induced gene silencing in tomato plants (TRV-SlMPK4) resulted in increased susceptibility to B. cinerea. Expression of defense-related genes SlPR1a and SlPR1b were up-regulated in the SlMPK4-silenced plants. Furthermore, silencing of the SlMPK4 gene also resulted in reduced tolerance against drought stress, leading to earlier wilting symptom under drought stress condition, as compared with the control plants. These results suggest important roles for SlMPK4 in disease resistance against B. cinerea and tolerance to drought stress.

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.

The de novo Biosynthesis of Vitamin B6 Is Required for Disease Resistance Against Botrytis cinerea in Tomato

Vitamin B6 (VB6), an essential cofactor for numerous metabolic enzymes, has recently been shown to act as a potent antioxidant and play important roles in developmental processes and stress responses. However, little is known about the possible function of VB6 in plant disease resistance response against pathogen infection. In the present study, we explored the possible involvement of VB6 in defense response against Botrytis cinerea through functional analysis of tomato VB6 biosynthetic genes. Three de novo VB6 biosynthetic genes (SlPDX1.2, SlPDX1.3, and SlPDX2) and one salvage pathway gene (SlSOS4) were identified and the SlPDX1.2, SlPDX1.3, and SlPDX2 genes were shown to encode functional enzymes involved in de novo biosynthesis of VB6, as revealed by complementation of the VB6 prototrophy in yeast snz1 and sno1 mutants. Expression of SlPDX1.2, SlPDX1.3, and SlSOS4 genes was induced by infection with B. cinerea. Virus-induced gene silencing-mediated knockdown of SlPDX1.2 or SlPDX1.3 but not SlPDX2 and SlSOS4 led to increased severity of disease caused by B. cinerea, indicating that the VB6 de novo biosynthetic pathway but not the salvage pathway is involved in tomato defense response against B. cinerea. Furthermore, the SlPDX1.2- and SlPDX1.3-silenced tomato plants exhibited reduced levels of VB6 contents and reactive oxygen species scavenging capability, increased levels of superoxide anion and H2O2 generation, and increased activity of superoxide dismutase after infection by B. cinerea. Our results suggest that VB6 and its de novo biosynthetic pathway play important roles in regulation of defense response against B. cinerea through modulating cellular antioxidant capacity.