Via Virus-induced Gene Silencing Research Articles

Molecular characterization of cassava zinc finger-homeodomain (ZF-HD) transcription factors reveals their role in disease resistance

The production of cassava (Manihot esculenta Crantz) is constantly threatened by cassava bacterial blight (CBB), caused by Xanthomonas phaseoli pv. manihotis (Xpm). Zinc finger-homeodomain (ZF-HD) belongs to a family of homozygous heterotypic cassette genes widely implicated in various developmental and physiological processes in plants. Despite their importance, a comprehensive analysis of ZF-HD genes, particularly those involved in disease resistance, has not been performed for cassava. In the present study, we utilized bioinformatics methods to identify 21 ZF-HD genes distributed across 11 chromosomes of cassava genome, with the majority exhibiting gene structure without introns. Phylogenetic analysis categorized these genes into two major groups (MIF and ZHD) with five subgroups. We observed fourteen pairs of duplicated genes, suggesting that segmental duplication has likely facilitated the expansion of the cassava ZF-HD gene family. Comparative orthologous analyses between cassava and other plant species shed light on the evolutionary trajectory of this gene family. Promoter analyses revealed multiple hormone- and stress-related elements, indicative of a functional role in stress responses. Expression profiling through RNA-seq and quantitative real-time PCR (qRT-PCR) demonstrated that certain cassava ZF-HD genes are up-regulated in response to Xpm infection, suggesting their involvement in defense mechanisms. Notably, MeZHD7 gene was identified via virus induced gene silencing (VIGS) as potentially crucial in conferring resistance against CBB. Results from subcellular localization experiments indicated that MeZHD7 was localized in the nucleus. The Luciferase reporter assay demonstrated an interaction between MeZHD7 and MeMIF5. These findings may lay the foundation for further cloning and functional analyses of cassava ZF-HD genes, particularly those associated with pathogen resistance.

Deciphering the role of SlWRKY36 and SlWRKY51 in salt stress tolerance via modulating ion homeostasis and proline biosynthesis

Soil salinity caused by NaCl is a major challenge to agricultural crops worldwide. For this, two WRKY transcription factors were evaluated for their role in salt stress tolerance in tomato plants (Solanum lycopersicum; Sl). SlWRKY36 and SlWRKY51 provided novel insight into the regulatory mechanism in tomato against salt stress via virus-induced gene silencing (VIGS). Salt stress significantly reduced chlorophyll-a, an abundant form of chlorophyll content to 6.0 and 5.1 mg/g and proline content to 0.06 mg/g and 0.09 mg/g respectively in SlWRKY36 and SlWRKY51 silenced tomato plants. This shows that salt stress affected proline content that act as osmo-protectant and damaged photosynthetic pigments in silenced SlWRKY36 and SlWRKY51 tomato plants. Similarly, the concentrations of Na+/ K+ ratio also showed a significantly higher trend 14 days after salt stress with 5.5 mg/g and 8.9 mg/g concentration at 200 mM for SlWRKY36 and SlWRKY51 showing silencing promotes Na+/K+ ion ratio under salt stress. Also, salt stress responsive genes such as salt overly sensitive SOS1 and Na+/H+ exchanger NHX1 displayed lower transcript level in silenced plants at 200 mM salt stress showing their negative regulation by SlWRKY36 and SlWRKY51 gene silencing. Collectively, these findings suggest for the first time the role of SlWRKY36 and SlWRKY51 as positive regulators of salt stress tolerance by managing ion homeostasis, proline content and photosynthetic machinery via transcriptional reprogramming. Overall, SlWRKY36 and SlWRKY51 were explored as potential candidates for engineering salt tolerance in tomato crop plants.

Comprehensive identification and systematical characterization of BRX gene family and the functional of GhBRXL5A in response to salt stress

BackgroundBRVIS RADIX (BRX) family is a small gene family with the highly conserved plant-specific BRX domains, which plays important roles in plant development and response to abiotic stress. Although BRX protein has been studied in other plants, the biological function of cotton BRX-like (BRXL) gene family is still elusive.ResultIn this study, a total of 36 BRXL genes were identified in four cotton species. Whole genome or segmental duplications played the main role in the expansion of GhBRXL gene family during evolutionary process in cotton. These BRXL genes were clustered into 2 groups, α and β, in which structural and functional conservation within same groups but divergence among different groups were found. Promoter analysis indicated that cis-elements were associated with the phytohormone regulatory networks and the response to abiotic stress. Transcriptomic analysis indicated that GhBRXL2A/2D and GhBRXL5A/5D were up/down-regulated in response to the different stress. Silencing of GhBRXL5A gene via virus-induced gene silencing (VIGS) improved salt tolerance in cotton plants. Furthermore, yeast two hybrid analysis suggested homotypic and heterotypic interactions between GhBRXL1A and GhBRXL5D.ConclusionsOverall, these results provide useful and valuable information for understanding the evolution of cotton GhBRXL genes and their functions in salt stress.

Genome-wide identification of SAMS gene family in Cucurbitaceae and the role of ClSAMS1 in watermelon tolerance to abiotic stress

S-Adenosyl-L-methionine (SAM) is widely involved in plant growth, development, and abiotic stress response. SAM synthetase (SAMS) is the key enzyme that catalyzes the synthesis of SAM from methionine and ATP. However, the SAMS gene family has not been identified and their functions have not been characterized in most Cucurbitaceae plants. Here, a total of 30 SAMS genes were identified in nine Cucurbitaceae species and they were categorized into 3 subfamilies. Physicochemical properties and gene structure analysis showed that the SAMS protein members are tightly conserved. Further analysis of the cis-regulatory elements (CREs) of SAMS genes' promoter implied their potential roles in stress tolerance. To further understand the molecular functions of SAMS genes, watermelon SAMSs (ClSAMSs) were chosen to analyze the expression patterns in different tissues and under various abiotic stress and hormone responses. Among the investigated genes, ClSAMS1 expression was observed in all tissues and found to be up-regulated by abiotic stresses including salt, cold and drought treatments as well as exogenous hormone treatments including ETH, SA, MeJA and ABA. Furthermore, knockdown of ClSAMS1 via virus-induced gene silencing (VIGS) decreased SAM contents in watermelon seedings. The pTRSV2-ClSAMS1 plants showed reduced susceptibility to drought, cold and NaCl stress, indicating a positive role of ClSAMS1 in abiotic stresses tolerance. Those results provided candidate SAMS genes to regulate plant resistance against abiotic stresses in Cucurbitaceae plants.

GhMAPK3, a mitogen-activated protein kinase, enhance salt and drought tolerance in cotton (Gossypium hirsutum)

Multiple abiotic stresses, such as drought and high salinity, seriously limit cotton production. However, the mitogen-activated protein kinases (MAPKs) could play a critical role in responding to multiple environmental abiotic stresses. In this study, one of the MAPKs in cotton, GhMAPK3 (GH_D03G1517), was cloned and its role in drought and salt stress tolerance was investigated. The qRT-PCR assays demonstrated that GhMAPK3 predominantly expressed in leaf and root parts and showed higher expression under salt and drought stress. Subcellular localization assays revealed that GhMAPK3 was distributed in the nucleus and cytoplasm. The silenced of GhMAPK3 could decrease the tolerance of cotton plants under salt and drought stress via virus-induced gene silencing (VIGS) experiments. In GhMAPK3-slienced plants, the expression of stress-responsive genes has been revealed to be down-regulated levels, while the oxidants and antioxidant enzymes showed relatively higher levels and relatively lower levels, respectively. We further acquired three GhMAPK3 overexpression Arabidopsis lines, and they showed higher drought and salt tolerance than the wild-type (WT) plants, the chlorophyll content and relative leaf water content were found to be relatively stable through GhMAPK3 overexpressed plants under the abiotic stress. Further investigation revealed that GhMAPK3 showed a higher germination rate and longer root length under salt and drought stress. Overall, our findings revealed that the GhMAPK3 gene plays a prominent role in drought and salt tolerance and offers knowledge on crop genetic improvements.

SmRAV1, an AP2 and B3 Transcription Factor, Positively Regulates Eggplant's Response to Salt Stress.

Salt stress is a lethal abiotic stress threatening global food security on a consistent basis. In this study, we identified an AP2 and B3 domain-containing transcription factor (TF) named SmRAV1, and its expression levels were significantly up-regulated by NaCl, abscisic acid (ABA), and hydrogen peroxide (H2O2) treatment. High expression of SmRAV1 was observed in the roots and sepal of mature plants. The transient expression assay in Nicotiana benthamiana leaves revealed that SmRAV1 was localized in the nucleus. Silencing of SmRAV1 via virus-induced gene silencing (VIGS) decreased the tolerance of eggplant to salt stress. Significant down-regulation of salt stress marker genes, including SmGSTU10 and SmNCED1, was observed. Additionally, increased H2O2 content and decreased catalase (CAT) enzyme activity were recorded in the SmRAV1-silenced plants compared to the TRV:00 plants. Our findings elucidate the functions of SmRAV1 and provide opportunities for generating salt-tolerant lines of eggplant.

Dynamic characteristics and functional analysis provide new insights into the role of GauERF105 for resistance against Verticillium dahliae in cotton

BackgroundThe cotton industry suffers significant yield losses annually due to Verticillium wilt, which is considered the most destructive disease affecting the crop. However, the precise mechanisms behind this disease in cotton remain largely unexplored.MethodsOur approach involved utilizing transcriptome data from G. australe which was exposed to Verticillium dahliae infection. From this data, we identified ethylene-responsive factors and further investigated their potential role in resistance through functional validations via Virus-induced gene silencing (VIGS) in cotton and overexpression in Arabidopsis.ResultsA total of 23 ethylene response factors (ERFs) were identified and their expression was analyzed at different time intervals (24 h, 48 h, and 72 h post-inoculation). Among them, GauERF105 was selected based on qRT-PCR expression analysis for further investigation. To demonstrate the significance of GauERF105, VIGS was utilized, revealing that suppressing GauERF105 leads to more severe infections in cotton plants compared to the wild-type. Additionally, the silenced plants exhibited reduced lignin deposition in the stems compared to the WT plants, indicating that the silencing of GauERF105 also impacts lignin content. The overexpression of GauERF105 in Arabidopsis confirmed its pivotal role in conferring resistance against Verticillium dahliae infection. Our results suggest that WT possesses higher levels of the oxidative stress markers MDA and H2O2 as compared to the overexpressed lines. In contrast, the activities of the antioxidant enzymes SOD and POD were higher in the overexpressed lines compared to the WT. Furthermore, DAB and trypan staining of the overexpressed lines suggested a greater impact of the disease in the wild-type compared to the transgenic lines.ConclusionsOur findings provide confirmation that GauERF105 is a crucial candidate in the defense mechanism of cotton against Verticillium dahliae invasion, and plays a pivotal role in this process. These results have the potential to facilitate the development of germplasm resistance in cotton.

GhCKX14 responding to drought stress by modulating antioxi-dative enzyme activity in Gossypium hirsutum compared to CKX family genes

BackgroundCytokinin oxidase/dehydrogenase (CKX) plays a vital role in response to abiotic stress through modulating the antioxidant enzyme activities. Nevertheless, the biological function of the CKX gene family has yet to be reported in cotton.ResultIn this study, a total of 27 GhCKXs were identified by the genome-wide investigation and distributed across 18 chromosomes. Phylogenetic tree analysis revealed that CKX genes were clustered into four clades, and most gene expansions originated from segmental duplications. The CKXs gene structure and motif analysis displayed remarkably well conserved among the four groups. Moreover, the cis-acting elements related to the abiotic stress, hormones, and light response were identified within the promoter regions of GhCKXs. Transcriptome data and RT-qPCR showed that GhCKX genes demonstrated higher expression levels in various tissues and were involved in cotton’s abiotic stress and phytohormone response. The protein-protein interaction network indicates that the CKX family probably participated in redox regulation, including oxidoreduction or ATP levels, to mediate plant growth and development. Functionally identified via virus-induced gene silencing (VIGS) found that the GhCKX14 gene improved drought resistance by modulating the antioxidant-related activitie.ConclusionsIn this study, the CKX gene family members were analyzed by bioinformatics, and validates the response of GhCKX gene to various phytohormone treatment and abiotic stresses. Our findings established the foundation of GhCKXs in responding to abiotic stress and GhCKX14 in regulating drought resistance in cotton.

The miR166d/TaCPK7-D Signaling Module Is a Critical Mediator of Wheat (Triticum aestivum L.) Tolerance to K+ Deficiency.

It is well established that potassium (K+) is an essential nutrient for wheat (Triticum aestivum L.) growth and development. Several microRNAs (miRNAs), including miR166, are reportedly vital roles related to plant growth and stress responses. In this study, a K+ starvation-responsive miRNA (miR166d) was identified, which showed increased expression in the roots of wheat seedlings exposed to low-K+ stress. The overexpression of miR166d considerably increased the tolerance of transgenic Arabidopsis plants to K+ deprivation treatment. Furthermore, disrupting miR166d expression via virus-induced gene silencing (VIGS) adversely affected wheat adaptation to low-K+ stress. Additionally, miR166d directly targeted the calcium-dependent protein kinase 7-D gene (TaCPK7-D) in wheat. The TaCPK7-D gene expression was decreased in wheat seedling roots following K+ starvation treatment. Silencing TaCPK7-D in wheat increased K+ uptake under K+ starvation. Moreover, we observed that the miR166d/TaCPK7-D module could affect wheat tolerance to K+ starvation stress by regulating TaAKT1 and TaHAK1 expression. Taken together, our results indicate that miR166d is vital for K+ uptake and K+ starvation tolerance of wheat via regulation of TaCPK7-D.

Identification of candidate genes for aphid resistance in upland cotton by QTL mapping and expression analysis

Lignin is one of the main components of cell walls and is essential for resistance to insect pests in plants. Cotton plants are damaged by aphid (Aphis gossypii) worldwide but resistant breeding is undeveloped due to scarce knowledge on resistance genes and the mechanism. This study reported a lignin biosynthesis-related gene identified in the F2 population derived from the cross between cotton cultivars Xinluzao 61 (resistant to aphid) and Xinluzao 50 (susceptible to aphid). A quantitative trait locus was mapped on chromosome D04 with a logarithm of odds (LOD) score of 5.99 and phenotypic effect of 27%. RNA-seq analysis of candidate intervals showed that the expression level of GH_D04G1418 was higher in the resistant cultivar than in the susceptible cultivar. This locus is close to AtLAC4 in the phylogenetic tree and contains a conserved laccase domain. Hence, it was designated GhLAC4-3. Silencing of GhLAC4-3 in Xinluzao 61 via virus-induced gene silencing (VIGS) resulted in decreased lignin content and increased susceptibility to aphids. These results suggest that GhLAC4-3 might enhance aphid resistance by regulating lignin biosynthesis in cotton.

Transcription factor CabHLH035 promotes cold resistance and homeostasis of reactive oxygen species in pepper

Plant basic helix-loop-helix (bHLH) transcription factors (TFs) play central roles in various abiotic stresses. However, its role in plant cold resistance is largely unknown. Previously, we characterised CaNAC035 in pepper, which positively regulates tolerance to cold, salt and drought stresses tolerance. Here, we identified CabHLH035, a CaNAC035-interacting protein in pepper. To explore its functions in cold stress tolerance, we silenced the gene in pepper via virus-induced gene silencing (VIGS) and overexpressed the gene in Arabidopsis. The results showed that CabHLH035 expression was induced by cold treatment, and silencing of CabHLH035 decreased cold stress tolerance. Conversely, overexpression of CabHLH035 in Arabidopsis increased cold stress tolerance. To investigate homologs genes of C-repeat binding factor (CBF) pathway proteins and reactive oxygen species (ROS) marker gene expression blocking by CabHLH035, we performed yeast one-hybrid (Y1H), dual luciferase and electrophoretic mobility shift assay experiments. The results showed that CabHLH035 bound to the region upstream of the CaCBF1A and CaAPX promoters. Additionally, CaCBF1A bound to the CaDHN4 promoter. Taken together, our results showed that CabHLH035 plays a crucial role in cold stress tolerance and its potential as a target for breeding cold-resistant crops. The findings provide a basis for studying the functions and regulatory network of cold stress tolerance in pepper.

Genome-wide association study reveals that GhTRL1 and GhPIN8 affect cotton root development.

Two regions located at chromosome A05 and D04 were found to be significantly associated with 0-0.5mm and 0.5-2mm diameter roots, respectively, and two candidate genes related to root development were identified. Roots absorb water and nutrients, and play an important role in plant growth. However, there are few genetic developmental studies on cotton root structural traits. In this study, we used 200 upland cotton (Gossypium hirsutum L.) varieties to analyze the phenotypic variation of 43 traits. A total of 2001 related single-nucleotide polymorphism (SNP) sites located within or near 1046 genes were detected through a genome-wide association study (GWAS). The 32 root traits were linked to SNPs that corresponded to 317 nonrepetitive genes. For SNPs associated with root length and 0-0.5mm diameter root traits, a significant peak appeared on chromosome A05 (between 21.91 and 22.24Mb). For SNPs associated with root surface area, root volume and 0.5-2mm diameter root traits, a significant peak appeared on chromosome D04 (between 7.35 and 7.70Mb). Within these two key regions, SNPs were detected in the promoter and coding regions of two candidate genes, GhTRL1-A05 and GhPIN8-D04. The expression levels of these two genes also changed significantly according to transcriptome sequencing and quantitative real-time PCR (qRT-PCR). After silencing the GhTRL1 and GhPIN8 genes via virus-induced gene silencing (VIGS), we found that the plants expressing TRV2::GhTRL1 and TRV2::GhPIN8 had a reduced root length, surface area. Moreover, the contents of cis-12-oxo-phytodienoic acid (cis-OPDA), isopentenyl adenosine (iPR) and cis-zeatin (cZ) in the roots of the plants expressing TRV2::GhTRL1 decreased. This study contributes to the cultivation and improvement of cotton varieties.

The regulation of Alternaria alternata resistance by LRR-RK4 through ERF109, defensin19 and phytoalexin scopoletin in Nicotiana attenuata

Leucine-rich repeat receptor-like kinases (LRR-RKs), belonging to the largest subfamily of transmembrane receptor-like kinases in plants, are proposed to be involved in pathogen resistance. However, it is currently unknown whether LRR-RKs regulate Nicotiana attenuata resistance to Alternaria alternata, a notorious fungal pathogen causing tobacco brown disease. During transcriptome analysis, we identified a highly induced receptor kinase (NaLRR-RK4) in N. attenuata leaves after A. alternata inoculation. We speculated that this NaLRR-RK4 might be the resistance gene of tobacco to brown spot disease, and if so, what is its function and mechanism of action? Silencing of NaLRR-RK4 via virus-induced gene silencing (VIGS) lead to plants highly susceptible to A. alternata, and this result was further confirmed by two stable transformation lines (NaLRR-RK4-RNAi lines) generated by RNA interference technology. The susceptible of NaLRR-RK4-RNAi lines to A. alternata was associated with reduced levels of phytoalexin scopoletin and its key synthesis gene NaF6′H1. Further transcriptome analysis of leaves of WT and NaLRR-RK4-RNAi line after A. alternata inoculation revealed that NaLRR-RK4 regulated NaERF109 and NaDEF19. Silencing NaERF109 or NaDEF19 by VIGS lead to plants more susceptible to A.alternata, demonstrating their role in pathogen resistance. Interestingly, A.alternata-induced expression of NaF6′H1 and NaDEF19 were dramatically reduced in NaERF109-silenced VIGS plants. Taken all together, we identified LRR-RK4 as the first Leucine-rich repeat receptor-like kinases involved in A.alternata resistance in tobacco species, by regulating NaERF109, and subsequently NaDEF19 and NaF6′H1.

GhHAI2, GhAHG3, and GhABI2 Negatively Regulate Osmotic Stress Tolerance via ABA-Dependent Pathway in Cotton (Gossypium hirsutum L.).

The type 2C protein phosphatases (PP2Cs) are well known for their vital roles in plant drought stress responses, but their molecular mechanisms in cotton (Gossypium hirsutum L.) remain largely unknown. Here, we investigated the role of three clade A PP2C genes, namely, GhHAI2, GhAHG3, and GhABI2, in regulating the osmotic stress tolerance in cotton. The transcript levels of GhHAI2, GhAHG3, and GhABI2 were rapidly induced by exogenous abscisic acid (ABA) and polyethylene glycol (PEG) treatment. Silencing of GhHAI2, GhAHG3, and GhABI2 via virus-induced gene silencing (VIGS) improved osmotic tolerance in cotton due to decreased water loss, increase in both relative water content (RWC) and photosynthetic gas exchange, higher antioxidant enzyme activity, and lower malondialdehyde (MDA) content. The root analysis further showed that GhHAI2, GhAHG3, and GhABI2-silenced plants were more responsive to osmotic stress. Yeast two-hybrid (Y2H) and luciferase complementation imaging (LCI) assays further substantiated that GhHAI2, GhAHG3, and GhABI2 interact with the core receptors of ABA signaling, GhPYLs. The expression of several ABA-dependent stress-responsive genes was significantly upregulated in GhHAI2-, GhAHG3-, and GhABI2-silenced plants. Our findings suggest that GhHAI2, GhAHG3, and GhABI2 act as negative regulators in the osmotic stress response in cotton through ABA-mediated signaling.

The Cassava NBS-LRR Genes Confer Resistance to Cassava Bacterial Blight.

Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) seriously affects cassava yield. Genes encoding nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains are among the most important disease resistance genes in plants that are specifically involved in the response to diverse pathogens. However, the in vivo roles of NBS-LRR remain unclear in cassava (Manihot esculenta). In this study, we isolated four MeLRR genes and assessed their expression under salicylic acid (SA) treatment and Xam inoculation. Four MeLRR genes positively regulate cassava disease general resistance against Xam via virus-induced gene silencing (VIGS) and transient overexpression. During cassava-Xam interaction, MeLRRs positively regulated endogenous SA and reactive oxygen species (ROS) accumulation and pathogenesis-related gene 1 (PR1) transcripts. Additionally, we revealed that MeLRRs positively regulated disease resistance in Arabidopsis. These pathogenic microorganisms include Pseudomonas syringae pv. tomato, Alternaria brassicicola, and Botrytis cinerea. Our findings shed light on the molecular mechanism underlying the regulation of cassava resistance against Xam inoculation.

A cysteine-rich receptor-like protein kinase CaCKR5 modulates immune response against Ralstonia solanacearum infection in pepper

BackgroundCysteine-rich receptor-like kinases (CRKs) represent a large subfamily of receptor-like kinases and play vital roles in diverse physiological processes in regulating plant growth and development.ResultsCaCRK5 transcripts were induced in pepper upon the infection of Ralstonia solanacearum and treatment with salicylic acid. The fusions between CaCRK5 and green fluorescence protein were targeted to the plasma membrane. Suppression of CaCRK5 via virus-induced gene silencing (VIGS) made pepper plants significantly susceptible to R. solanacearum infection, which was accompanied with decreased expression of defense related genes CaPR1, CaSAR8.2, CaDEF1 and CaACO1. Overexpression of CaCRK5 increased resistance against R. solanacearum in Nicotiana benthamiana. Furthermore, electrophoretic mobility shift assay and chromatin immunoprecipitation coupled with quantitative real-time PCR analysis revealed that a homeodomain zipper I protein CaHDZ27 can active the expression of CaCRK5 through directly binding to its promoter. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses suggested that CaCRK5 heterodimerized with the homologous member CaCRK6 on the plasma membrane.ConclusionsOur data revealed that CaCRK5 played a positive role in regulating immune responses against R. solanacearum infection in pepper.

Systematical characterization of YUCCA gene family in five cotton species, and potential functions of YUCCA22 gene in drought resistance of cotton

Auxin plays an essential role in the normal growth and development of plants. The tryptophan aminotransferase of Arabidopsis (TAA)/YUCCA (YUC) enzymes functions as flavin monooxygenases (FMOs), which are the key enzymes in auxin synthesis. However, comprehensive characterization of the YUC gene family in cotton has not yet been completed. In order to reveal the functions of YUC genes in cotton, we systematically identified and characterized the YUC gene family in five species of cotton including analysis of the member, characterization, intragenomic duplication, and collinear relationships. Transcriptomic analysis indicates that GhYUCs participate in abiotic stress processes in cotton. The GhYUC22 gene that is responsive to polyethylene glycol (PEG) and abscisic acid (ABA) was functionally identified via virus-induced gene silencing (VIGS). Subsequent natural drought simulation experiments and physiological index data demonstrated that partial silencing of GhYUC22 gene endowed cotton with drought resistance by modulating indole-3-acetic acid (IAA) and ABA signaling pathways. In conclusion, our findings support the role of that GhYUC22 gene in modulating both endogenous auxin and ABA homeostasis and regulating the drought resistance in cotton.

TaClpS1, negatively regulates wheat resistance against Puccinia striiformis f. sp. tritici

BackgroundThe degradation of intracellular proteins plays an essential role in plant responses to stressful environments. ClpS1 and E3 ubiquitin ligase function as adaptors for selecting target substrates in caseinolytic peptidase (Clp) proteases pathways and the 26S proteasome system, respectively. Currently, the role of E3 ubiquitin ligase in the plant immune response to pathogens is well defined. However, the role of ClpS1 in the plant immune response to pathogens remains unknown.ResultsHere, wheat (Triticum aestivum) ClpS1 (TaClpS1) was studied and resulted to encode 161 amino acids, containing a conserved ClpS domain and a chloroplast transit peptide (1–32 aa). TaClpS1 was found to be specifically localized in the chloroplast when expressed transiently in wheat protoplasts. The transcript level of TaClpS1 in wheat was significantly induced during infection by Puccinia striiformis f. sp. tritici (Pst). Knockdown of TaClpS1 via virus-induced gene silencing (VIGS) resulted in an increase in wheat resistance against Pst, accompanied by an increase in the hypersensitive response (HR), accumulation of reactive oxygen species (ROS) and expression of TaPR1 and TaPR2, and a reduction in the number of haustoria, length of infection hypha and infection area of Pst. Furthermore, heterologous expression of TaClpS1 in Nicotiana benthamiana enhanced the infection by Phytophthora parasitica.ConclusionsThese results suggest that TaClpS1 negatively regulates the resistance of wheat to Pst.

The G-protein α subunit GhGPA positively regulates Gossypium hirsutum resistance to Verticillium dahliae via induction of SA and JA signaling pathways and ROS accumulation

Verticillium wilt, a devastating disease in cotton caused by Verticillium dahliae, reduces cotton quality and yield. Heterotrimeric GTP-binding proteins, consisting of Gα, Gβ, and Gγ subunits, transducers of receptor signaling, function in a wide range of biological events. However, the function of Gα proteins in the regulation of defense responses in plants is largely unexplored, except for a few reports on model species. In the present study, a cotton G-protein α-subunit-encoding gene (GhGPA) was isolated from Verticillium wilt-resistant Gossypium hirsutum (upland cotton) cv. ND601. GhGPA transcription was up-regulated under V. dahliae stress, with higher expression in tolerant than in susceptible cotton cultivars. Subcellular localization revealed GhGPA to be located in the plasma membrane. GhGPA shows high (85.0%) identity with Arabidopsis AT2G26300 (AtGPA1), and AtGPA1 gpa1-4 mutants displayed susceptibility to V. dahliae. Ectopic expression of GhGPA successfully restored the resistance of Arabidopsis gpa1-4 mutants to Verticillium wilt and made them more resistant than the wild type. Overexpression of GhGPA in Arabidopsis markedly increased the resistance and resulted in dramatic up-regulation of pathogenesis-related (PR) genes and increased in H2O2 accumulation and salicylic acid (SA) and jasmonic acid (JA) contents. However, suppressing GhGPA expression via virus-induced gene silencing (VIGS) increased susceptibility to Verticillium wilt, down-regulated the expression of PR and marker genes in SA and JA signaling pathways, and reduced H2O2 content. The contents of SA and JA in Arabidopsis gpa1-4 and VIGS cotton were lower than those in the wild type and empty-vector control. However, GhGPA-overexpressing Arabidopsis contained more SA and JA than the wild type when inoculated with V. dahliae. Thus, GhGPA plays a vital role in Verticillium wilt resistance by inducing SA and JA signaling pathways and regulating the production of reactive oxygen species. These findings not only broaden our knowledge about the biological role of GhGPA, but also shed light on the defense mechanisms involving GhGPA against V. dahliae in cotton.

StRac1 plays an important role in potato resistance against Phytophthora infestans via regulating H2O2 production

ROP GTPases (Rho-related GTPases from plant), a unique subgroup of the Rho family in plants, is a group of key regulators of different signaling pathways controlling plant growth and development, cell polarity and differentiation, and plant response against biotic and abiotic stresses. The present study determined the potential regulatory mechanism of potato ROP GTPase (StRac1) against Phytophthora infestans (P. infestans) infection. Protein secondary structure analysis indicated that StRAC1 is a Rho GTPase. The expression level of StRac1 was variable in different tissues of potato, with the highest expression in young leaves of both Shepody and Hutou potato varieties. After challenging with P. infestans, the expression level of StRac1was higher in resistance varieties Zihuabai and Longshu 7 than in susceptible varieties Shepody and Desiree. StRAC1 fusion with GFP subcellularly localized at the plasma membrane (PM) in tobacco epidermal cells. The potato with transient or stable over-expression of CA-StRac1 (constitutively active form of StRac1)exhibited a dramatic enhancement of its resistance against P. infestans infections. The increased resistance level in transgenic potato was accompanied with elevated H2O2 levels. Importantly, silencing StRac1 via virus-induced gene silencing (VIGS) in potato resulted in higher susceptibility to P. infestans infection than in control plants. In summary, our data reveal that StRac1 regulates potato resistance against P. infestans via positively modulating the accumulation of H2O2.