Hypersensitive Response-like Cell Death Research Articles

CaARP1/CaSGT1 Module Regulates Vegetative Growth and Defense Response of Pepper Plants against Phytophthora capsici

Pepper (Capsicum annuum L.) suffers severe quality and yield loss from oomycete diseases caused by Phytophthora capsici. CaSGT1 was previously determined to positively regulate the immune response of pepper plants against P. capsici, but by which mechanism remains elusive. In the present study, the potential interacting proteins of CaSGT1 were isolated from pepper using a yeast two-hybrid system, among which CaARP1 was determined to interact with CaSGT1 via bimolecular fluorescence complementation (BiFC) and microscale thermophoresis (MST) assays. CaARP1 belongs to the auxin-repressed protein family, which is well-known to function in modulating plant growth. The transcriptional and protein levels of CaARP1 were both significantly induced by infection with P. capsici. Silencing of CaARP1 promotes the vegetative growth of pepper plants and attenuates its disease resistance to P. capsici, as well as compromising the hypersensitive response-like cell death in pepper leaves induced by PcINF1, a well-characterized typical PAMP from P. capsici. Chitin-induced transient expression of CaARP1 in pepper leaves enhanced its disease resistance to P. capsici, which is amplified by CaSGT1 co-expression as a positive regulator. Taken together, our result revealed that CaARP1 plays a dual role in the pepper, negatively regulating the vegetative growth and positively regulating plant immunity against P. capsici in a manner associated with CaSGT1.

CaWRKY22b Plays a Positive Role in the Regulation of Pepper Resistance to Ralstonia solanacearum in a Manner Associated with Jasmonic Acid Signaling.

As important transcription factors, WRKYs play a vital role in the defense response of plants against the invasion of multiple pathogens. Though some WRKY members have been reported to participate in pepper immunity in response to Ralstonia solanacearum infection, the functions of the majority of WRKY members are still unknown. Herein, CaWRKY22b was cloned from the pepper genome and its function against R. solanacearum was analyzed. The transcript abundance of CaWRKY22b was significantly increased in response to the infection of R. solanacearum and the application of exogenous methyl jasmonate (MeJA). Subcellular localization assay in the leaves of Nicotiana benthamiana showed that CaWRKY22b protein was targeted to the nuclei. Agrobacterium-mediated transient expression in pepper leaves indicated that CaWRKY22b overexpression triggered intensive hypersensitive response-like cell death, H2O2 accumulation, and the up-regulation of defense- and JA-responsive genes, including CaHIR1, CaPO2, CaBPR1, and CaDEF1. Virus-induced gene silencing assay revealed that knock-down of CaWRKY22b attenuated pepper's resistance against R. solanacearum and the up-regulation of the tested defense- and jasmonic acid (JA)-responsive genes. We further assessed the role of CaWRKY22b in modulating the expression of JA-responsive CaDEF1, and the result demonstrated that CaWRKY22b trans-activated CaDEF1 expression by directly binding to its upstream promoter. Collectively, our results suggest that CaWRKY22b positively regulated pepper immunity against R. solanacearum in a manner associated with JA signaling, probably by modulating the expression of JA-responsive CaDEF1.

CaMAPK1 Plays a Vital Role in the Regulation of Resistance to Ralstonia solanacearum Infection and Tolerance to Heat Stress.

As an important member of mitogen-activated protein kinase (MAPK) cascades, MAPKs play an important role in plant defense response against biotic and abiotic stresses; however, the involvement of the majority of the MAPK family members against Ralstonia solanacearum and heat stress (HS) remains poorly understood. In the present study, CaMAPK1 was identified from the genome of pepper and its function against R. solanacearum and HS was analyzed. The transcript accumulations of CaMAPK1 and the activities of its native promoter were both significantly induced by R. solanacearum inoculation, HS, and the application of exogenous hormones, including SA, MeJA, and ABA. Transient expression of CaMAPK1 showed that CaMAPK1 can be targeted throughout the whole cells in Nicotiana benthamiana and triggered chlorosis and hypersensitive response-like cell death in pepper leaves, accompanied by the accumulation of H2O2, and the up-regulations of hormones- and H2O2-associated marker genes. The knock-down of CaMAPK1 enhanced the susceptibility to R. solanacearum partially by down-regulating the expression of hormones- and H2O2-related genes and impairing the thermotolerance of pepper probably by attenuating CaHSFA2 and CaHSP70-1 transcripts. Taken together, our results revealed that CaMAPK1 is regulated by SA, JA, and ABA signaling and coordinates responses to R. solanacearum infection and HS in pepper.

Effect of RNAi suppression of the gossypol pathway on resistance to necrotrophic fungal seedling disease pathogens in cotton

We previously demonstrated that blocking the gossypol pathway by RNAi suppression of the early pathway biosynthetic enzyme CYP82D hydroxylase resulted in enhanced resistance to the hemibiotrophic Fusarium wilt pathogens. The enhanced resistance was attributed to the induction of a linked lacinilene defense pathway. Others reported that RNAi suppression of the same gene, which was named as SILENCE-INDUCED STEM NECROSIS (SSN), caused spontaneous hypersensitive response-like cell death resulting in a lesion mimic phenotype and increased resistance to hemibiotrophic Verticillium wilt pathogens. We observed no lesion formation on hypocotyls when the CYP82D RNAi plants were grown in pasteurized soil at warm temperatures that are unfavorable to seedling disease development. Therefore, RNAi suppression of CYP82D is not associated with spontaneous hypersensitive-like cell death. When grown in unpasteurized soil at cool temperatures that are favorable for seedling disease development, more than 87% of the CYP82D RNAi plants developed necrotic hypocotyl lesions, compared to 3% of the wild type plants that developed lesions. Furthermore, at least 83% of the necrotic CYP82D RNAi plants harbored at least one of the isolates of Fusarium spp, Rhizopus spp., or Aspergillus spp. that are often implicated in the seedling diseases of cotton. Pathogenicity assays with the seedling disease pathogen Rhizoctonia solani AG4 confirmed that blocking the gossypol defense pathway by CYP82D RNAi increased host susceptibility to the necrotrophic pathogen. The concentrations of induced lacinilene products detected in the roots and hypocotyls of RNAi plants when treated with Rhizoctonia were far lower than the concentrations seen previously in these tissues induced by Fusarium wilt pathogens. Thus, the increased susceptibility of the RNAi plants to Rhizoctonia was probably due to the combined effect of low concentrations of lacinilenes and the blockage of the gossypol pathway by the RNAi. Developing cotton host resistance to wilt diseases without compromising resistance to seedling disease pathogens requires further understanding of the interaction between signaling pathways involved in responses to hemibiotrophs and necrotrophs.

A ubiquitin-specific protease functions in regulating cell death and immune responses in rice.

Ubiquitin-specific proteases (UBPs) process deubiquitination in eukaryotic organisms and are widely involved in plant development and responses to environmental stress. However, their role in cell death and plant immunity remains largely unknown. Here, we identified a rice lesion mimic mutant (LMM) and cloned its causative gene, LMM22. Both dysfunction and overexpression of LMM22 gave rise to the hypersensitive response-like cell death, reactive oxygen species bursts, and activated defence responses. LMM22 encodes an active UBP that is localised to the endoplasmic reticulum (ER) and displays a constitutive expression pattern in rice. LMM22 interacts with SPOTTED LEAF 35 (SPL35), a coupling of ubiquitin conjugation to ER degradation domain-containing protein that is known to participate in ubiquitination and the regulation of cell death and disease response in rice. Additional analyses suggest that LMM22 can positively regulate and stabilise the abundance of SPL35 protein likely through its deubiquitination activity. These data therefore improve our understanding of the function of UBP in rice innate immune responses by demonstrating that LMM22 functions as a critical regulator of SPL35 in cell death and disease resistance.

ATP-citrate lyase B (ACLB) negatively affects cell death and resistance to Verticillium wilt

BackgroundATP-citrate lyase (ACL) plays a pivotal role in histone acetylation and aerobic glycolysis. In plant, ACL is a heteromeric enzyme composed of ACLA (45 kD) and ACLB (65 kD). So far, the function of ACL genes in cotton still remains unknown.ResultsHere, we identified three ACLA homologous sequences and two ACLB homologous in each genome/sub-genome of cotton species. Silencing ACLB in cotton led to cell death at newly-grown leaves and stem apexes. Simultaneously, in ACLB-silenced plants, transcription factors related to senescence including SGR, WRKY23 and Osl57 were observed to be activated. Further investigation showed that excessive H2O2 was accumulated, salicylic acid-dependent defense response and pathogenesis-related gene expressions were evidently enhanced in ACLB-silenced plants, implying that knockdown of ACLB genes leads to hypersensitive response-like cell death in cotton seedlings. However, as noted, serious cell death happened in newly-grown leaves and stem apexes in ACLB-silenced plants, which led to the failure of subsequent fungal pathogenicity assays. To confirm the role of ACLB gene in regulating plant immune response, the dicotyledonous model plant Arabidopsis was selected for functional verification of ACLB gene. Our results indicate the resistance to Verticillium dahliae infection in the Arabidopsis mutant aclb-2 were enhanced without causing strong cell death. Ectopic expression of GausACLB-2 in Arabidopsis weakened its resistance to V. dahliae either in Col-0 or in aclb-2 background, in which the expression level of ACLB is negatively correlated with the resistance to V. dahliae.ConclusionsThese results indicate that ACLB has a new function in negatively affecting the induction of plant defense response and cell death in cotton, which provides theoretical guidance for developing cotton varieties with resistance against Verticillium wilt.

Deletion of Diterpenoid Biosynthetic Genes CYP76M7 and CYP76M8 Induces Cell Death and Enhances Bacterial Blight Resistance in Indica Rice '9311'.

Lesion mimic mutants (LMMs) are ideal materials for studying cell death and resistance mechanisms. Here, we identified and mapped a novel rice LMM, g380. The g380 exhibits a spontaneous hypersensitive response-like cell death phenotype accompanied by excessive accumulation of reactive oxygen species (ROS) and upregulated expression of pathogenesis-related genes, as well as enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo). Using a map-based cloning strategy, a 184,916 bp deletion on chromosome 2 that overlaps with the diterpenoid biosynthetic gene cluster was identified in g380. Accordingly, the content of diterpenoids decreased in g380. In addition, lignin, one of the physical lines of plant defense, was increased in g380. RNA-seq analysis showed 590 significantly differentially expressed genes (DEG) between the wild-type 9311 and g380, 585 of which were upregulated in g380. Upregulated genes in g380 were mainly enriched in the monolignol biosynthesis branches of the phenylpropanoid biosynthesis pathway, the plant–pathogen interaction pathway and the phytoalexin-specialized diterpenoid biosynthesis pathway. Taken together, our results indicate that the diterpenoid biosynthetic gene cluster on chromosome 2 is involved in immune reprogramming, which in turn regulates cell death in rice.

Genetic analysis reveals three novel QTLs underpinning a butterfly egg-induced hypersensitive response-like cell death in Brassica rapa.

BackgroundCabbage white butterflies (Pieris spp.) can be severe pests of Brassica crops such as Chinese cabbage, Pak choi (Brassica rapa) or cabbages (B. oleracea). Eggs of Pieris spp. can induce a hypersensitive response-like (HR-like) cell death which reduces egg survival in the wild black mustard (B. nigra). Unravelling the genetic basis of this egg-killing trait in Brassica crops could improve crop resistance to herbivory, reducing major crop losses and pesticides use. Here we investigated the genetic architecture of a HR-like cell death induced by P. brassicae eggs in B. rapa.ResultsA germplasm screening of 56 B. rapa accessions, representing the genetic and geographical diversity of a B. rapa core collection, showed phenotypic variation for cell death. An image-based phenotyping protocol was developed to accurately measure size of HR-like cell death and was then used to identify two accessions that consistently showed weak (R-o-18) or strong cell death response (L58). Screening of 160 RILs derived from these two accessions resulted in three novel QTLs for Pieris brassicae-induced cell death on chromosomes A02 (Pbc1), A03 (Pbc2), and A06 (Pbc3). The three QTLs Pbc1–3 contain cell surface receptors, intracellular receptors and other genes involved in plant immunity processes, such as ROS accumulation and cell death formation. Synteny analysis with A. thaliana suggested that Pbc1 and Pbc2 are novel QTLs associated with this trait, while Pbc3 also contains an ortholog of LecRK-I.1, a gene of A. thaliana previously associated with cell death induced by a P. brassicae egg extract.ConclusionsThis study provides the first genomic regions associated with the Pieris egg-induced HR-like cell death in a Brassica crop species. It is a step closer towards unravelling the genetic basis of an egg-killing crop resistance trait, paving the way for breeders to further fine-map and validate candidate genes.

Hypersensitive response-like cell death and its key related genes in thelmdlesion mimic mutant of birch

Lesion mimic mutants (LMMs) often display hypersensitive response-like (HR-like) cell death and enhanced resistance to pathogens. LMMs have been viewed as useful for the study of the mechanism of cell death and plant immunity. To date, few LMMs have been found in woody plants. We previously identified an LMM termed lmd, which showed spontaneous cell death in birch. In the current study, we investigated lmd, oe21 (transgenic control), and NT (nontransgenic control), focusing on cell death and gene expression profile of lmd. We found that cell death in the lmd occurred gradually during leaf development. The number of necrotic spots increased as the leaf developed, with no significant change in spot size. Subcellular observations showed degradation of both mitochondria and chloroplasts in lmd cells but not in NT and oe21 cells. Autophagosomes were visible in dying cells of lmd. We then performed RNA-seq of the four most apical leaves of lmd and oe21 to explore the gene expression profile during cell death. Genes that were functional in signal perception, respiratory burst, signal transduction, and defense were enriched. TGA1, WRKY33, WRKY40, Chitinase, FLS2, RbohA/D, and SBT were identified as key genes involved in cell death in lmd.

Universal gene co-expression network reveals receptor-like protein genes involved in broad-spectrum resistance in pepper (Capsicum annuum L.).

Receptor-like proteins (RLPs) on plant cells have been implicated in immune responses and developmental processes. Although hundreds of RLP genes have been identified in plants, only a few RLPs have been functionally characterized in a limited number of plant species. Here, we identified RLPs in the pepper (Capsicum annuum) genome and performed comparative transcriptomics coupled with the analysis of conserved gene co-expression networks (GCNs) to reveal the role of core RLP regulators in pepper–pathogen interactions. A total of 102 RNA-seq datasets of pepper plants infected with four pathogens were used to construct CaRLP-targeted GCNs (CaRLP-GCNs). Resistance-responsive CaRLP-GCNs were merged to construct a universal GCN. Fourteen hub CaRLPs, tightly connected with defense-related gene clusters, were identified in eight modules. Based on the CaRLP-GCNs, we evaluated whether hub CaRLPs in the universal GCN are involved in the biotic stress response. Of the nine hub CaRLPs tested by virus-induced gene silencing, three genes (CaRLP264, CaRLP277, and CaRLP351) showed defense suppression with less hypersensitive response-like cell death in race-specific and non-host resistance response to viruses and bacteria, respectively, and consistently enhanced susceptibility to Ralstonia solanacearum and/or Phytophthora capsici. These data suggest that key CaRLPs are involved in the defense response to multiple biotic stresses and can be used to engineer a plant with broad-spectrum resistance. Together, our data show that generating a universal GCN using comprehensive transcriptome datasets can provide important clues to uncover genes involved in various biological processes.

A conserved clathrin-coated vesicle component, OsSCYL2, regulates plant innate immunity in rice.

Clathrin‐mediated vesicle trafficking (CMVT) is a fundamental process in all eukaryotic species, and indispensable to organism's growth and development. Recently, it has been suggested that CMVT also plays important roles in the regulation of plant immunity. However, the molecular link between CMVT and plant immunity is largely unknown. SCY1‐LIKE2 (SCYL2) is evolutionally conserved among the eukaryote species. Loss‐of‐function of SCYL2 in Arabidopsis led to severe growth defects. Here, we show that mutation of OsSCYL2 in rice gave rise to a novel phenotype—hypersensitive response‐like (HR) cell death in a light‐dependent manner. Although mutants of OsSCYL2 showed additional defects in the photosynthetic system, they exhibited enhanced resistance to bacterial pathogens. Subcellular localisation showed that OsSCYL2 localized at Golgi, trans‐Golgi network and prevacuolar compartment. OsSCYL2 interacted with OsSPL28, subunit of a clathrin‐associated adaptor protein that is known to regulate HR‐like cell death in rice. We further showed that OsSCYL2–OsSPL28 interaction is mediated by OsCHC1. Collectively, we characterized a novel component of the CMVT pathway in the regulation of plant immunity. Our work also revealed unidentified new functions of the very conserved SCYL2. It thus may provide new breeding targets to achieve both high yield and enhanced resistance in crops.

Rapeseed NAC46 positively regulates hypersensitive response-like cell death and chlorophyll degradation

Plants are frequently challenged by various stresses, such as abiotic and biotic stresses. The NAC (NAM/ATAF/CUC) transcription factor family is specific to plants and play diverse roles including resistance to abiotic and biotic stresses, while the functions of many rapeseed (Brassica napus L.) NACs are still unknown. In this study, we identified that rapeseed NAC46 positively regulated hypersensitive response (HR) -like cell death. BnaNAC46 expression was induced by multiple stress and phytohormone treatments and was highly expressed in senescent leaves. BnaNAC46 localized in the nucleus and showed transcriptional activation activity in both yeasts and plant cells. BnaNAC46 bound to a cis-element of NACBS (NAC binding sequence). Overexpression of BnaNAC46 resulted in HR-like cell death and chlorophyll degradation. Results of dual luciferase, gel shift and chromatin immunoprecipitation coupled to qPCR assays have demonstrated that BnaNAC46 directly bound to promoters of BnaBFN1, BnaCEP1, BnaMC9, BnaγVPE, BnaNYE1 and BnaPPH genes implicated in programmed cell death and chlorophyll degradation, via the NACBS element. In summary, this study has revealed that rapeseed NAC46 is a transcriptional activator that acts as a positive regulator of cell death and chlorophyll degradation.

The role of chaperone complex HSP90-SGT1-RAR1 as the associated machinery for hybrid inviability between Nicotiana gossei Domin and N. tabacum L.

Two cultured cell lines (GTH4 and GTH4S) of a Nicotiana interspecific F1 hybrid (N. gossei×N. tabacum) were comparatively analyzed to find genetic factors related to hybrid inviability. Both cell lines proliferated at 37°C, but after shifting to 26°C, GTH4 started to die similar to the F1 hybrid seedlings, whereas GTH4S survived. As cell death requires de novo expression of genes and proteins, we compared expressed protein profiles between the two cell lines, and found that NgSGT1, a cochaperone of the chaperone complex (HSP90-SGT1-RAR1), was expressed in GTH4 but not in GTH4S. Agrobacterium-mediated transient expression of NgSGT1, but not NtSGT1, induced cell death in leaves of N. tabacum, suggesting its possible role in hybrid inviability. Cell death in N. tabacum was also induced by transient expression of NgRAR1, but not NtRAR1. In contrast, transient expression of any parental combinations of three components revealed that NgRAR1 promoted cell death, whereas NtRAR1 suppressed it in N. tabacum. A specific inhibitor of HSP90, geldanamycin, inhibited the progression of hypersensitive response-like cell death in GTH4 and leaf tissue after agroinfiltration. The present study suggested that components of the chaperone complex are involved in the inviability of Nicotiana interspecific hybrid.

Characterization of Pathogenicity-Associated V2 Protein of Tobacco Curly Shoot Virus.

V2 proteins encoded by some whitefly-transmitted geminiviruses were reported to be functionally important proteins. However, the functions of the V2 protein of tobacco curly shoot virus (TbCSV), a monopartite begomovirus that causes leaf curl disease on tomato and tobacco in China, remains to be characterized. In our report, an Agrobacterium infiltration-mediated transient expression assay indicated that TbCSV V2 can suppress local and systemic RNA silencing and the deletion analyses demonstrated that the amino acid region 1–92 of V2, including the five predicted α-helices, are required for local RNA silencing suppression. Site-directed substitutions showed that the conserved basic and ring-structured amino acids in TbCSV V2 are critical for its suppressor activity. Potato virus X-mediated heteroexpression of TbCSV V2 in Nicotiana benthamiana induced hypersensitive response-like (HR-like) cell death and systemic necrosis in a manner independent of V2′s suppressor activity. Furthermore, TbCSV infectious clone mutant with untranslated V2 protein (TbCSV∆V2) could not induce visual symptoms, and coinfection with betasatellite (TbCSB) could obviously elevate the viral accumulation and symptom development. Interestingly, symptom recovery occurred at 15 days postinoculation (dpi) and onward in TbCSV∆V2/TbCSB-inoculated plants. The presented work contributes to understanding the RNA silencing suppression activity of TbCSV V2 and extends our knowledge of the multifunctional role of begomovirus-encoded V2 proteins during viral infections.

A Histopathological Study Reveals New Insights Into Responses of Chestnut (Castanea spp.) to Root Infection by Phytophthora cinnamomi.

The European chestnut (Castanea sativa) is threatened by the hemibiotrophic oomycete Phytophthora cinnamomi, the causal agent of ink disease. Chestnut species have different susceptibility levels to P. cinnamomi, with the Asian species (C. crenata; C. mollissima) exhibiting the highest level of resistance. A histological approach was used to study the responses exhibited by susceptible and resistant chestnut genotypes by characterizing the early stages of P. cinnamomi infection and the cellular responses it induces in roots. C. sativa (susceptible) and C. crenata (resistant) plantlets were inoculated with a P. cinnamomi virulent isolate with a zoospore suspension or by direct contact with mycelia agar pieces. Root samples were collected at 0.5, 3.5, 24, 48, and 72 h after inoculation (hai) for microscopic observations. Penetration was observed in both species at 0.5 and 3.5 hai with mycelium and zoospore inoculations, respectively. In both inoculation methods, following penetration into the rhizodermis, P. cinnamomi hyphae grew inter- and intracellularly through the cortex and into the vascular cylinder. C. crenata cells displayed a delay in the pattern of infection by having fewer cell layers colonized compared with C. sativa. At 72 hai, the collapse of the first layers of C. sativa cortical cells was observed, indicating the beginning of necrotrophy. C. crenata was able to respond more efficiently to P. cinnamomi than C. sativa by restricting the pathogen's growth area through the early activation of resistance responses such as callose deposition around some intracellular hyphae, hypersensitive response-like cell death, cell wall thickening, and accumulation of phenolic-like compounds.

Ectopic overexpression of a membrane-tethered transcription factor gene NAC60 from oilseed rape positively modulates programmed cell death and age-triggered leaf senescence.

Senescence is an integrative final stage of plant development that is governed by internal and external cues. The NAM, ATAF1/2, CUC2 (NAC) transcription factor (TF) family is specific to plants and membrane-tethered NAC TFs (MTTFs) constitute a unique and sophisticated mechanism in stress responses and development. However, the function of MTTFs in oilseed rape (Brassica napus L.) remains unknown. Here, we report that BnaNAC60 is an MTTF associated with the endoplasmic reticulum (ER) membrane. Expression of BnaNAC60 was induced during the progression of leaf senescence. Translocation of BnaNAC60 into nuclei was induced by ER stress and oxidative stress treatments. It binds to the NTLBS motif, rather than the canonical NAC recognition site. Overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, induces significant reactive oxygen species (ROS) accumulation and hypersensitive response-like cell death in both tobacco (Nicotiana benthamiana) and oilseed rape protoplasts. Moreover, ectopic overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, in Arabidopsis also induces precocious leaf senescence. Furthermore, screening and expression profiling identified an array of functional genes that are significantly induced by BnaNAC60 expression. Further it was found that BnaNAC60 can activate the promoter activities of BnaNYC1, BnaRbohD, BnaBFN1, BnaZAT12, and multiple BnaVPEs in a dual-luciferase reporter assay. Electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative PCR assays revealed that BnaNAC60 directly binds to the promoter regions of these downstream target genes. To summarize, our data show that BnaNAC60 is an MTTF that modulates cell death, ROS accumulation, and leaf senescence.

CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew

BackgroundThe development of CRISPR/Cas9 technology has facilitated targeted mutagenesis in an efficient and precise way. Previously, RNAi silencing of the susceptibility (S) gene PowderyMildewResistance 4 (PMR4) in tomato has been shown to enhance resistance against the powdery mildew pathogen Oidium neolycopersici (On).ResultsTo study whether full knock-out of the tomato PMR4 gene would result in a higher level of resistance than in the RNAi-silenced transgenic plants we generated tomato PMR4 CRISPR mutants. We used a CRISPR/Cas9 construct containing four single-guide RNAs (sgRNAs) targeting the tomato PMR4 gene to increase the possibility of large deletions in the mutants. After PCR-based selection and sequencing of transformants, we identified five different mutation events, including deletions from 4 to 900-bp, a 1-bp insertion and a 892-bp inversion. These mutants all showed reduced susceptibility to On based on visual scoring of disease symptoms and quantification of relative fungal biomass. Histological observations revealed a significantly higher occurrence of hypersensitive response-like cell death at sites of fungal infection in the pmr4 mutants compared to wild-type plants. Both haustorial formation and hyphal growth were diminished but not completely inhibited in the mutants.ConclusionCRISPR/Cas-9 targeted mutagenesis of the tomato PMR4 gene resulted in mutants with reduced but not complete loss of susceptibility to the PM pathogen On. Our study demonstrates the efficiency and versatility of the CRISPR/Cas9 system as a powerful tool to study and characterize S-genes by generating different types of mutations.

Citron C-05 inhibits both the penetration and colonization of Xanthomonas citri subsp. citri to achieve resistance to citrus canker disease

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a serious bacterial disease that affects citrus production worldwide. Citron C-05 (Citrus medica) is the only germplasm in the Citrus genus that has been identified to exhibit strong resistance to Xcc. However, it has not been determined when, where, and how Xcc is restricted in the tissues of Citron C-05 during the infection process. In the present study, we investigated the spatiotemporal growth dynamics of an eGFP-labeled virulent Xcc (eGFP-Xcc) strain in Citron C-05 along with five susceptible biotypes (i.e., lemon, pummelo, sour orange, sweet orange, and ponkan mandarin) upon inoculation via the spraying or leaf infiltration of a bacterial suspension. The results from extensive confocal laser scanning microscopy analyses showed that while Xcc grew rapidly in plants of all five susceptible genotypes, Xcc was severely restricted in the epidermal and mesophyll cell layers of the leaves of Citron C-05 in the early stage of infection. Not surprisingly, resistance against Xcc in Citron C-05 was found to be associated with the production of reactive oxygen species and hypersensitive response-like cell death, as well as greater upregulation of several defense-related genes, including a pathogenesis-related gene (PR1) and a glutathione S-transferase gene (GST1), compared with sweet orange as a susceptible control. Taken together, our results not only provide further valuable details of the spatiotemporal dynamics of the host entry, propagation, and spread of Xcc in both resistant and susceptible citrus plants but also suggest that resistance to Xcc in Citron C-05 may be attributed to the activation of multiple defense mechanisms.

The R2R3-MYB transcription factor BnaMYB111L from rapeseed modulates reactive oxygen species accumulation and hypersensitive-like cell death

As one of the largest families of transcription factors in plants, the R2R3-MYB proteins play important roles in diverse biological processes including growth and development, primary and secondary metabolism such as flavonoid and anthocyanin biosynthesis as well as abiotic and biotic stress responses. However, functions of R2R3-MYB genes in rapeseed (Brassica napus L.) remain elusive. Here, we characterized BnaMYB111L, which is homologous to Arabidopsis MYB111 and encodes an R2R3-MYB protein in rapeseed. BnaMYB111L is responsive to abscisic acid (ABA), heat, cold, hydrogen peroxide and fungal pathogen Sclerotinia scelerotiorum treatments through quantitative RT-PCR assay. BnaMYB111L encodes a transcriptional activator and is localized exclusively to nuclei. Interestingly, overexpression of BnaMYB111L in tobacco (Nicotiana benthamiana) and rapeseed protoplasts promoted reactive oxygen species (ROS) production and hypersensitive response-like cell death, accumulation of malondialdehyde (MDA) as well as degradation of chlorophyll. Furthermore, BnaMYB111L expression evoked the alterations of transcript levels of genes encoding ROS-producing enzyme, vacuolar processing enzymes and genes implicated in defense responses. A further dual luciferase reporter assay indicated that BnaMYB111L activated the expression of RbohB, MC4 and ACRE132, which are involved in ROS generation, cell death as well as defense responses. Taken together, this study characterized the function of rapeseed MYB111L and identified its putative target genes involved in ROS production and cell death.

The cytological basis of powdery mildew resistance in wild Chinese Vitis species

The wild Chinese grapevines (Vitis spp.) show varying levels of resistance to powdery mildew caused by Erysiphe necator that is an economically important disease of cultivated grapevines (Vitis vinifera). However, little information is available regarding the cytological mechanisms of powdery mildew resistance in these wild relatives. Here, we studied the cytological responses of three wild Chinese grapevine accessions after they were infected with E. necator (En) NAFU1 in comparison to the susceptible V. vinifera cv. ‘Thompson Seedless’ grape. The hyphal growth and sporulation of En NAFU1 were significantly restricted in wild species compared to ‘Thompson Seedless’, which appears to be associated with early cell wall deposition at the attempt sites, encasement of haustoria, and hypersensitive response-like cell death of penetrated epidermal cells. Moreover, endogenous free salicylic acid (SA) was more abundant in wild Chinese Vitis species than in ‘Thompson Seedless’ under pathogen-free condition. During En NAFU1 colonization, SA conjugates accumulated higher in wild grapevines than in ‘Thompson Seedless’. In addition, the species-specific expression patterns of defense-associated genes during En NAFU1 colonization indicated that mechanisms underlying powdery mildew resistance are divergent among different wild Chinese Vitis species. These results contribute to understanding of mechanisms underlying defense responses of wild Chinese Vitis species against powdery mildew.