Detection and Quantification of Candidatus Methanoperedens-Like Archaea in Freshwater Wetland Soils.

Candidatus Methanoperedens-like archaea, which can use multiple electron acceptors (nitrate, iron, manganese, and sulfate) for anaerobic methane oxidation, could play an important role in reducing methane emissions from freshwater wetlands. Currently, very little is known about the distribution and community composition of Methanoperedens-like archaea in freshwater wetlands, particularly based on their alpha subunit of methyl-coenzyme M reductase (mcrA) genes. Here, the community composition, diversity, and abundance of Methanoperedens-like archaea were investigated in a freshwater wetland through high-throughput sequencing and quantitative PCR on their mcrA genes. A large number of Methanoperedens-like mcrA gene sequences (119,250) were recovered, and a total of 31 operational taxonomic units (OTUs) were generated based on 95% sequence similarity cut-off. The majority of Methanoperedens-like sequences can be grouped into three distinct clusters that were closely associated with the known Methanoperedens species which can couple anaerobic methane oxidation to nitrate or iron reduction. The community composition of Methanoperedens-like archaea differed significantly among different sampling sites, and their mcrA gene abundance was 1.49 × 106 ~ 4.62 × 106 copies g-1 dry soil in the examined wetland. In addition, the community composition of Methanoperedens-like archaea was significantly affected by the soil water content, and the archaeal abundance was significantly positively correlated with the water content. Our results suggest that the mcrA gene is a good biomarker for detection and quantification of Methanoperedens-like archaea, and provide new insights into the distribution and environmental regulation of these archaea in freshwater wetlands.

Influence of affinity tags and tobacco PR1a signal peptide on detection, purification and bioactivity analyses of the small oomycete apoplastic effectors.

OBJECTIVE: To examine the influence of widely used protein affinity tags and the tobacco PR1a signal peptide (SP) on detection, purification and bioactivity analyses of the small oomycete apoplastic effector SCR96 in planta. RESULTS: Through agroinfiltration, the phytotoxic effector SCR96 of Phytophthora cactorum was expressed in Nicotiana benthamiana leaf apoplast as a fusion protein carrying single affinity tag (His, HA or FLAG) at either C- or N-terminus. Leaf necrosis caused by different affinity-tagged SCR96 varied among tags and replicates. All of tagged proteins can be detected by antibodies against SCR96. All of SCR96 fusions except N-terminally fused 6His-tagged protein were detected using tag antibodies, indicating that 6His tag may be degraded when fused at N-terminus. Interestingly, C-terminal His- and FLAG-tagged SCR96 maintained the biological activity after purification. In the substitution assay of SCR96 SP, we observed that PR1a SP can lead chimeric SCR96 expression in N. benthamiana, but the replacement totally disrupted its bioactivity. CONCLUSION: C-terminal His or FLAG tag, along with its original SP, is efficient enough to enable detection and purification of functional SCR96 from N. benthamiana leaf apoplast, which would facilitate plant-pathogen interaction studies.

Effect of gradual increase of atmospheric CO2 concentration on nitrification potential and communities of ammonia oxidizers in paddy fields.

Currently, the influence of elevated atmospheric CO2 concentration (eCO2) on ammonia oxidation to nitrite, the rate-limiting step of nitrification in paddy soil, is poorly known. Previous studies that simulate the effect of eCO2 on nitrification are primarily based on an abrupt increase of atmospheric CO2 concentration. However, paddy ecosystems are experiencing a gradual increase of CO2 concentration. To better understand how the nitrification potential, abundance and communities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) respond to eCO2 in paddy ecosystems, a field experiment was conducted using the following two treatments: a gradual increase of CO2 (EC, increase of 40 ppm per year until 200 ppm above ambient) and ambient CO2 (CK). The results demonstrated that the EC treatment significantly (P < 0.05) stimulated the soil potential nitrification rate (PNR) at the jointing and milky stages, which increased by 127.83% and 27.35%, respectively, compared with CK. Furthermore, the EC treatment significantly (P < 0.05) stimulated the AOA and AOB abundance by 56.60% and 133.84%, respectively, at the jointing stage. Correlation analysis showed that the PNR correlated well with the abundance of AOB (R2 = 0.7389, P < 0.001). In addition, the EC treatment significantly (P < 0.05) altered the community structure of AOB, while it had little effect on that of AOA. A significant difference in the proportion of Nitrosospira was observed between CO2 treatments. In conclusion, the gradual increase of CO2 positively influenced the PNR and abundance of ammonia oxidizers, and AOB could be more important than AOA in nitrification under eCO2.

ACT001 inhibits pituitary tumor growth by inducing autophagic cell death via MEK4/MAPK pathway.

ACT001, derived from traditional herbal medicine, is a novel compound with effective anticancer activity in clinical trials. However, little is known regarding its role in pituitary adenomas. Here, we demonstrated that ACT001 suppressed cell proliferation and induced cell death of pituitary tumor cells in vitro and in vivo. ACT001 was also effective in suppressing the growth of different subtypes of human pituitary adenomas. The cytotoxic mechanism ACT001 employed was mainly related to autophagic cell death (ACD), indicated by autophagosome formation and LC3-II accumulation. In addition, ACT001-mediated inhibitory effect decreased when either ATG7 was downregulated or cells were cotreated with autophagy inhibitor 3-methyladenine (3-MA). RNA-seq analysis showed that mitogen-activated protein kinase (MAPK) pathway was a putative target of ACT001. Specifically, ACT001 treatment promoted the phosphorylation of JNK and P38 by binding to mitogen-activated protein kinase kinase 4 (MEK4). Our study indicated that ACT001-induced ACD of pituitary tumor cells via activating JNK and P38 phosphorylation by binding with MEK4, and it might be a novel and effective anticancer drug for pituitary adenomas.

A Small Cysteine-Rich Phytotoxic Protein of Phytophthora capsici Functions as Both Plant Defense Elicitor and Virulence Factor.

Small cysteine-rich (SCR) proteins, including fungal avirulence proteins, play important roles in pathogen-plant interactions. SCR protein-encoding genes have been discovered in the genomes of Phytophthora pathogens but their functions during pathogenesis remain obscure. Here, we report the characterization of one Phytophthora capsici SCR protein (namely, SCR82) with similarity to Phytophthora cactorum phytotoxic protein PcF. The scr82 gene has 10 allelic sequences in the P. capsici population. Homologs of SCR82 were not identified in fungi or other organisms but in Phytophthora relative species. Initially, scr82 was weakly expressed during the mycelium, sporangium, and zoospore stages but quickly upregulated when the infection initiated. Both ectopic expression of SCR82 and recombinant yeast-expressed protein (rSCR82) caused cell death on tomato leaves. Upon treatment, rSCR82 induced plant defense responses, including the induction of defense gene expression, reactive oxygen species burst, and callose deposition. Knockout of scr82 in P. capsici by CRISPR/Cas9 severely impaired its virulence on host plants and significantly reduced its resistance against oxidative stress. Inversely, its overexpression increased the pathogen's virulence and tolerance to oxidative stress. Our results collectively demonstrate that SCR82 functions as both an important virulence factor and plant defense elicitor, which is conserved across Phytophthora spp.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

CaSBP11 Participates in the Defense Response of Pepper to Phytophthora capsici through Regulating the Expression of Defense-Related Genes.

Squamosa promoter binding protein (SBP)-box genes are plant-specific transcription factors involved in plant growth and development, morphogenesis and biotic and abiotic stress responses. However, these genes have been understudied in pepper, especially with respect to defense responses to Phytophthora capsici infection. CaSBP11 is a SBP-box family gene in pepper that was identified in our previous research. Silencing CaSBP11 enhanced the defense response of pepper plants to Phytophthora capsici. Without treatment, the expression of defense-related genes (CaBPR1, CaPO1, CaSAR8.2 and CaDEF1) increased in CaSBP11-silenced plants. However, the expression levels of these genes were inhibited under transient CaSBP11 expression. CaSBP11 overexpression in transgenic Nicotiana benthamiana decreased defense responses, while in Arabidopsis, it induced or inhibited the expression of genes in the salicylic acid and jasmonic acid signaling pathways. CaSBP11 overexpression in sid2-2 mutants induced AtNPR1, AtNPR3, AtNPR4, AtPAD4, AtEDS1, AtEDS5, AtMPK4 and AtNDR1 expression, while AtSARD1 and AtTGA6 expression was inhibited. CaSBP11 overexpression in coi1-21 and coi1-22 mutants, respectively, inhibited AtPDF1.2 expression and induced AtPR1 expression. These results indicate CaSBP11 has a negative regulatory effect on defense responses to Phytophthora capsici. Moreover, it may participate in the defense response of pepper to Phytophthora capsici by regulating defense-related genes and the salicylic and jasmonic acid-mediated disease resistance signaling pathways.

Transcription Factor CaSBP12 Negatively Regulates Salt Stress Tolerance in Pepper (Capsicum annuum L.).

SBP-box (Squamosa-promoter binding protein) genes are a type of plant-specific transcription factor and play important roles in plant growth, signal transduction, and stress response. However, little is known about the role of pepper SBP-box transcription factor genes in response to abiotic stress. Here, one of the pepper SBP-box gene, CaSBP12, was selected and isolated from pepper genome database in our previous study. The CaSBP12 gene was induced under salt stress. Silencing the CaSBP12 gene enhanced pepper plant tolerance to salt stress. The accumulation of reactive oxygen species (ROS) of the detached leaves of CaSBP12-silenced plants was significantly lower than that of control plants. Besides, the Na+, malondialdehyde content, and conductivity were significantly increased in control plants than that in the CaSBP12-silenced plants. In addition, the CaSBP12 over-expressed Nicotiana benthamiana plants were more susceptible to salt stress with higher damage severity index percentage and accumulation of ROS as compared to the wild-type. These results indicated that CaSBP12 negatively regulates salt stress tolerance in pepper may relate to ROS signaling cascades.

Genome-wide identification of the AP2/ERF transcription factor family in pepper (Capsicum annuum L.).

The AP2/ERF family is one of the largest transcription factor families in the plant kingdom. AP2/ERF genes contributing to various processes including plant growth, development, and response to various stresses have been identified. In this study, 175 putative AP2/ERF genes were identified in the latest pepper genome database and classified into AP2, RAV, ERF, and Soloist subfamilies. Their chromosomal localization, gene structure, conserved motif, cis-acting elements within the promoter region, and subcellular locations were analyzed. Transient expression of CaAP2/ERF proteins in tobacco revealed that CaAP2/ERF064, CaAP2/ERF109, and CaAP2/ERF127 were located in the nucleus, while CaAP2/ERF171 was located in the nucleus and cytoplasm. Most of the CaAP2/ERF genes contained cis-elements within their promoter regions that responded to various stresses (HSE, LTR, MBS, Box-W1/W-box, and TC-rich repeats) and phytohormones (ABRE, CGTCA-motif, and TCA-element). Furthermore, RNA-seq analysis revealed that CaAP2/ERF genes showed differential expression profiles in various tissues as well as under biotic stresses. Moreover, qRT-PCR analysis of eight selected CaAP2/ERF genes also showed differential expression patterns in response to infection with Phytophthora capsici (HX-9) and in response to phytohormones (SA, MeJA, and ETH). This study will provide basic insights for further studies of the CaAP2/ERF genes involved in the interaction between pepper and P. capsici.

A Novel Transcription Factor CaSBP12 Gene Negatively Regulates the Defense Response against Phytophthora capsici in Pepper (Capsicum annuum L.).

SBP-box (Squamosa-promoter binding protein) genes are a type of plant-specific transcription factor and play important roles in plant growth, signal transduction and stress response. However, little is known about the SBP-box genes in pepper (CaSBP), especially in the process of Phytophthora capsici infection. In this study, a novel gene (CaSBP12) was selected from the CaSBP gene family, which was isolated from the pepper genome database in our previous study. The CaSBP12 gene was located in the nucleus of the cell and its silencing in the pepper plant enhanced the defense response against Phytophthora capsici infection. After inoculation with Phytophthora capsici, the root activity of the CaSBP12-silenced plants is compared to control plants, while malondialdehyde (MDA) content is compared viceversa. Additionally, the expression of defense related genes (CaPO1, CaSAR8.2, CaBPR1, and CaDEF1) in the silenced plants were induced to different degrees and the peak of CaSAR8.2 and CaBPR1 were higher than that of CaDEF1. The CaSBP12 over-expressed Nicotiana benthamiana plants were more susceptible to Phytophthora capsici infection with higher EC (electrical conductivity) and MDA contents as compared to the wild-type. The relative expression of defense related genes (NbDEF, NbNPR1, NbPR1a, and NbPR1b) in transgenic and wild-type Nicotiana benthamiana plants were induced, especially the NbPR1a and NbPR1b. In conclusion, these results indicate that CaSBP12 gene negative regulates the defense response against Phytophthora capsici infection which suggests their potentially significant role in plant defense. To our knowledge, this is the first report on CaSBP gene which negative regulate defense response.

Time-Course Transcriptome Profiling Reveals Differential Resistance Responses of Tomato to a Phytotoxic Effector of the Pathogenic Oomycete Phytophthora cactorum.

Blight caused by Phytophthora pathogens has a devastating impact on crop production. Phytophthora species secrete an array of effectors, such as Phytophthora cactorum-Fragaria (PcF)/small cysteine-rich (SCR) phytotoxic proteins, to facilitate their infections. Understanding host responses to such proteins is essential to developing next-generation crop resistance. Our previous work identified a small, 8.1 kDa protein, SCR96, as an important virulence factor in Phytophthora cactorum. Host responses to SCR96 remain obscure. Here, we analyzed the effect of SCR96 on the resistance of tomato treated with this recombinant protein purified from yeast cells. A temporal transcriptome analysis of tomato leaves infiltrated with 500 nM SCR96 for 0, 3, 6, and 12 h was performed using RNA-Seq. In total, 36,779 genes, including 2704 novel ones, were detected, of which 32,640 (88.7%) were annotated. As a whole, 5929 non-redundant genes were found to be significantly co-upregulated in SCR96-treated leaves (3, 6, 12 h) compared to the control (0 h). The combination of annotation, enrichment, and clustering analyses showed significant changes in expression beginning at 3 h after treatment in genes associated with defense and metabolism pathways, as well as temporal transcriptional accumulation patterns. Noticeably, the expression levels of resistance-related genes encoding receptor-like kinases/proteins, resistance proteins, mitogen-activated protein kinases (MAPKs), transcription factors, pathogenesis-related proteins, and transport proteins were significantly affected by SCR96. Quantitative reverse transcription PCR (qRT-PCR) validated the transcript changes in the 12 selected genes. Our analysis provides novel information that can help delineate the molecular mechanism and components of plant responses to effectors, which will be useful for the development of resistant crops.

The devastating oomycete phytopathogen Phytophthora cactorum: Insights into its biology and molecular features.

Phytophthora cactorum is one of the most economically important soilborne oomycete pathogens in the world. It infects more than 200 plant species spanning 54 families, most of which are herbaceous and woody species. Although traditionally considered to be a generalist, marked differences of P. cactorum isolates occur in degree of pathogenicity to different hosts. As the impact of crop loss caused by this species has increased recently, there has been a tremendous increase in the development of new tools, resources, and management strategies to study and combat this devastating pathogen. This review aims to integrate recent molecular biology analyses of P. cactorum with the current knowledge of the cellular and genetic basis of its growth, development, and host infection. The goal is to provide a framework for further studies of P. cactorum by highlighting important biological and molecular features, shedding light on the functions of pathogenicity factors, and developing effective control measures. TAXONOMY: P. cactorum (Leb. & Cohn) Schröeter: kingdom Chromista; phylum Oomycota; class Oomycetes; order Peronosporales; family Peronosporaceae; genus Phytophthora. HOST RANGE: Infects about 200 plant species in 154 genera representing 54 families. Economically important host plants include strawberry, apple, pear, Panax spp., and walnut. DISEASE SYMPTOMS: The soilborne pathogen often causes root, stem, collar, crown, and fruit rots, as well as foliar infection, stem canker, and seedling damping off.

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Paddy fields are one of the most important methane sources, which have great impacts on climate change. The nitrite-dependent anaerobic methane oxidation, by NC10 phylum bacteria-Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria, is a new process regulating methane emission from paddy fields. However, little is known about the spatial and temporal variations of M. oxyfera-like bacterial communities and the regulating factors in paddy soils. We investigated the community composition, diversity, and abundance of M. oxyfera-like bacteria in 0-40 cm depth of paddy soils at key growth stages of rice, including tillering, jointing, flowering, and milky stages. Results of high-throughput sequencing showed that community composition of M. oxyfera-like bacteria differed significantly among different soil layers, while no significant variation was observed among different rice growth stages. The diversity of M. oxyfera-like bacteria increased with soil depth. Real-time quantitative PCR showed that the 16S rRNA gene abundance of M. oxyfera-like bacteria ranged from 5.73×106 to 2.56×107 copies·g-1 (dry weight), with the highest gene abundance in the 10-20 cm layer. Further, the abundance of these bacteria showed a decreasing trend with rice growth. Soil organic carbon content and soil pH were correlated with the M. oxyfera-like bacterial community structures and abundance. In all, our results suggested a certain degree of heterogeneity of spatial and temporal distribution of M. oxyfera-like bacterial communities in paddy soils, which was largely influenced by soil organic carbon and soil pH.

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Nitrate-dependent anaerobic oxidation of methane (AOM) is a new pathway to reduce methane emissions from paddy ecosystems. The elevated atmospheric CO2 concentration can affect methane emissions from paddy ecosystems, but its impact on the process of nitrate-dependent AOM is poorly known. Based on the automatic CO2 control platform with open top chambers and the 13CH4 stable isotope experiments, the responses of the activity of nitrate-dependent AOM, abundance and community composition of Candidatus Methanoperedens nitroreducens (M. nitroreducens)-like archaea to the gradual increase of CO2 concentration were investigated in paddy fields. We set up two CO2 concentration treatments, including an ambient CO2 and a gradual increase of CO2(increase of 40 µL·L-1 per year above ambient CO2 concentration until 160 µL·L-1). The results showed the nitrate-dependent AOM rate of 0.7-11.3 nmol CO2·g-1·d-1 in the studied paddy fields, and quantitative PCR showed the abundance of M. nitroreducens-like archaeal mcrA genes of 2.2×106-8.5×106 copies·g-1. Compared to the ambient CO2 treatment, the slow elevated CO2 treatment enhanced the nitrate-dependent AOM rate and stimulated the abundance of M. nitroreducens-like archaea, particularly in 5-10 cm soil layer. The gradual increased CO2 concentration treatment did not change the community composition of M. nitroreducens-like archaea, but significantly decreased their diversity. The soil organic carbon content was an important factor influencing the nitrate-dependent AOM process. Overall, our results showed that the gradual increase of CO2 concentration could promote the nitrate-dependent AOM, suggesting its positive role in mitigating methane emissions from paddy ecosystems under future climate change.

Different responses of ammonia-oxidizing archaea and bacteria in paddy soils to elevated CO2 concentration.

The elevated atmospheric CO2 concentration is well known to have an important effect on soil nutrient cycling. Ammonia oxidation, mediated by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), is the rate-limiting step in soil nitrification, which controls the availability of two key soil nutrients (ammonium and nitrate) for crops. Until now, how the AOA and AOB communities in paddy soils respond to elevated CO2 remains largely unknown. Here, we examined the communities of AOA and AOB and nitrification potential at both surface (0-5 cm) and subsurface (5-10 cm) soil layers of paddy fields under three different CO2 treatments, including CK (ambient CO2 concentration), LT (CK + 160 ppm of CO2) and HT (CK + 200 ppm of CO2). The elevated CO2 was found to have a greater impact on the community structure of AOB than that of AOA in surface soils as revealed by high-throughput sequencing of their amoA genes. However, no obvious variation of AOA or AOB communities was observed in subsurface soils among different CO2 treatments. The abundance of AOA and AOB, and nitrification potential were significantly increased in surface soils under elevated CO2. The variation of AOB abundance correlated well with the variation of nitrification potential. The soil water content and dissolved organic carbon content had important impacts on the dynamic of AOB communities and nitrification potential. Overall, our results showed different responses of AOA and AOB communities to elevated CO2 in paddy ecosystems, and AOB were more sensitive to the rising CO2 concentration.

Response of nitrite-dependent anaerobic methanotrophs to elevated atmospheric CO2 concentration in paddy fields.

Nitrite-dependent anaerobic methane oxidation (n-damo) catalyzed by Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria is a new pathway for the regulation of methane emissions from paddy fields. Elevated atmospheric CO2 concentrations (e[CO2]) can indirectly affect the structure and function of microbial communities. However, the response of M. oxyfera-like bacteria to e[CO2] is currently unknown. Here, we investigated the effect of e[CO2] (ambient CO2 + 200 ppm) on community composition, abundance, and activity of M. oxyfera-like bacteria at different depths (0-5, 5-10, and 10-20 cm) in paddy fields across multiple rice growth stages (tillering, jointing, and flowering). High-throughput sequencing showed that e[CO2] had no significant effect on the community composition of M. oxyfera-like bacteria. However, quantitative PCR suggested that the 16S rRNA gene abundance of M. oxyfera-like bacteria increased significantly in soil under e[CO2], particularly at the tillering stage. Furthermore, 13CH4 tracer experiments showed potential n-damo activity of 0.31-8.91 nmol CO2 g-1 (dry soil) d-1. E[CO2] significantly stimulated n-damo activity, especially at the jointing and flowering stages. The n-damo activity and abundance of M. oxyfera-like bacteria increased by an average of 90.9% and 50.0%, respectively, under e[CO2]. Correlation analysis showed that the increase in soil dissolved organic carbon content caused by e[CO2] had significant effects on the activity and abundance of M. oxyfera-like bacteria. Overall, this study provides the first evidence for a positive response of M. oxyfera-like bacteria to e[CO2], which may help reduce methane emissions from paddy fields under future climate change conditions.

Identification of Pepper CaSBP08 Gene in Defense Response Against Phytophthora capsici Infection.

Little information is available on the role of Squamosa promoter binding protein (SBP)-box genes in pepper plants. This family of genes is known to have transcription characteristics specific to plants and to regulate plant growth, development, stress responses, and signal transduction. To investigate their specific effects in pepper (Capsicum annuum), we screened pepper SBP-box family genes (CaSBP genes) for Phytophthora capsici (P. capsici) resistance genes using virus-induced gene silencing. CaSBP08, CaSBP11, CaSBP12, and CaSBP13, which are associated with plant defense responses against P. capsici, were obtained from among fifteen identified CaSBP genes. The function of CaSBP08 was identified in pepper defense response against P. capsici infection in particular. CaSBP08 protein was localized to the nucleus. Silencing of CaSBP08 enhanced resistance to P. capsici infection. Following P. capsici inoculation, the malondialdehyde content, peroxidase activity, and disease index percentage of the CaSBP08-silenced plants decreased compared to the control. Additionally, the expression levels of other defense-related genes, especially those of CaBPR1 and CaSAR8.2, were more strongly induced in CaSBP08-silenced plants than in the control. However, CaSBP08 overexpression in Nicotiana benthamiana enhanced susceptibility to P. capsici infection. This work provides a foundation for the further research on the role of CaSBP genes in plant defense responses against P. capsici infection.

The CaAP2/ERF064 Regulates Dual Functions in Pepper: Plant Cell Death and Resistance to Phytophthora capsici.

Phytophthora blight is one of the most destructive diseases of pepper (Capsicum annuum L.) globally. The APETALA2/Ethylene Responsive Factors (AP2/ERF) genes play a crucial role in plant response to biotic stresses but, to date, have not been studied in the context of Phytophthora resistance in pepper. Here, we documented potential roles for the pepper CaAP2/ERF064 gene in inducing cell death and conferring resistance to Phytophthora capsici (P. capsici) infection. Results revealed that the N-terminal, AP2 domain, and C-terminal of CaAP2/ERF064 protein is responsible for triggering cell death in Nicotiana benthamiana (N. benthamiana). Moreover, the transcription of CaAP2/ERF064 in plant is synergistically regulated by the Methyl-Jasmonate (MeJA) and ethephon (ET) signaling pathway. CaAP2/ERF064 was found to regulate the expression of CaBPR1, which is a pathogenesis-related (PR) gene of pepper. Furthermore, the silencing of CaAP2/ERF064 compromised the pepper plant resistance to P.capsici by reducing the transcript level of defense-related genes CaBPR1, CaPO2, and CaSAR82, while the ectopic expression of CaAP2/ERF064 in N. benthamiana plant elevated the expression level of NbPR1b and enhanced resistance to P.capsici. These results suggest that CaAP2/ERF064 could positively regulate the defense response against P. capsici by modulating the transcription of PR genes in the plant.

Genome-wide analysis of dirigent gene family in pepper (Capsicum annuum L.) and characterization of CaDIR7 in biotic and abiotic stresses.

The dirigent (DIR and DIR-like) proteins involved in lignification, play a pivotal role against biotic and abiotic stresses in plants. However, no information is available about DIR gene family in pepper (Capsicum annuum L.). In this study, 24 putative dirigent genes (CaDIRs) were identified, their gene structure, genome location, gene duplication and phylogenetic relationship were elucidated. Tissue-specific expression analysis displayed the highest transcription levels in flower, stem and leaf. Some CaDIRs were up-regulated by virulent (CaDIR2, 3, 6, 7, 11, 14, 16, 22 and 23) and avirulent (CaDIR3, 5, 7, 16, 20, 22, 23 and 24) Phytophthora capsici strains, as well as by Methyl jasmonate, salicylic acid, NaCl and mannitol stresses. Acid-soluble lignin content increased (103.21%) after P. capsici inoculation (48-hour). Silencing of CaDIR7 weakened plant defense by reducing (~50%) root activity and made plants more susceptible (35.7%) to P. capsici and NaCl (300 mM). Leaf discs of the CaDIR7:silenced plants exposed to NaCl and mannitol (300 mM each), exhibited a significant decrease (56.25% and 48% respectively) in the chlorophyll content. These results suggested that CaDIR7 is involved in pepper defense response against pathogen and abiotic stresses and the study will provide basic insights for future research regarding CaDIRs.

Genome-Wide Identification and Analysis of the SBP-Box Family Genes under Phytophthora capsici Stress in Pepper (Capsicum annuum L.).

SQUAMOSA promoter binding protein (SBP)-box genes encode plant-specific transcription factors that are extensively involved in many physiological and biochemical processes, including growth, development, and signal transduction. However, pepper (Capsicum annuum L.) SBP-box family genes have not been well characterized. We investigated SBP-box family genes in the pepper genome and characterized these genes across both compatible and incompatible strain of Phytophthora capsici, and also under different hormone treatments. The results indicated that total 15 members were identified and distributed on seven chromosomes of pepper. Phylogenetic analysis showed that SBP-box genes of pepper can be classified into six groups. In addition, duplication analysis within pepper genome, as well as between pepper and Arabidopsis genomes demonstrated that there are four pairs of homology of SBP-box genes in the pepper genome and 10 pairs between pepper and Arabidopsis genomes. Tissue-specific expression analysis of the CaSBP genes demonstrated their diverse spatiotemporal expression patterns. The expression profiles were similarly analyzed following exposure to P. capsici inoculation and hormone treatments. It was shown that nine of the CaSBP genes (CaSBP01, 02, 03, 04, 05, 06, 11, 12, and 13) exhibited a dramatic up-regulation after compatible HX-9 strain (P. capsici) inoculation, while CaSBP09 and CaSBP15 were down-regulated. In case of PC strain (P. capsici) infection six of the CaSBP genes (CaSBP02, 05, 06, 11, 12, and 13) were arose while CaSBP14 was down regulated. Furthermore, Salicylic acid, Methyl jasmonate and their biosynthesis inhibitors treatment indicated that some of the CaSBP genes are potentially involved in these hormone regulation pathways. This genome-wide identification, as well as characterization of evolutionary relationships and expression profiles of the pepper CaSBP genes, will help to improve pepper stress tolerance in the future.

VIGS approach reveals the modulation of anthocyanin biosynthetic genes by CaMYB in chili pepper leaves.

The purple coloration of pepper leaves arises from the accumulation of anthocyanin. Three regulatory and 12 structural genes have been characterized for their involvement in the anthocyanin biosynthesis. Examination of the abundance of these genes in leaves showed that the majority of them differed between anthocyanin pigmented line Z1 and non-pigmented line A3. Silencing of the R2R3-MYB transcription factor CaMYB in pepper leaves of Z1 resulted in the loss of anthocyanin accumulation. Moreover, the expression of multiple genes was altered in the silenced leaves. The expression of MYC was significantly lower in CaMYB-silenced leaves, whereas WD40 showed the opposite pattern. Most structural genes including CHS, CHI, F3H, F3'5'H, DFR, ANS, UFGT, ANP, and GST were repressed in CaMYB-silenced foliage with the exception of PAL, C4H, and 4CL. These results indicated that MYB plays an important role in the regulation of anthocyanin biosynthetic related genes. Besides CaMYB silenced leaves rendered more sporulation of Phytophthora capsici Leonian indicating that CaMYB might be involved in the defense response to pathogens.