scholarly journals Agromonas: a rapid disease assay for Pseudomonas syringae growth in agroinfiltrated leaves

2020 ◽  
Author(s):  
Pierre Buscaill ◽  
Nattapong Sanguankiattichai ◽  
Yoon Joo Lee ◽  
Jiorgos Kourelis ◽  
Gail Preston ◽  
...  
Keyword(s):  
Transient Expression ◽  
Pseudomonas Syringae ◽  
Nicotiana Benthamiana ◽  
Function Analysis ◽  
Plant Immunity ◽  
Comprehensive Analysis ◽  
Immune Gene ◽  
Proof Of Concept ◽  
Easy Method ◽  
Lengthy Process

SUMMARYThe lengthy process to generate transformed plants is a limitation in current research on the interactions of the model plant pathogen Pseudomonas syringae with plant hosts. Here we present an easy method called agromonas, where we quantify P. syringae growth in agroinfiltrated leaves of Nicotiana benthamiana using a cocktail of antibiotics to select P. syringae on plates. As a proof of concept, we demonstrate that transient expression of PAMP receptors reduces bacterial growth and that transient depletion of a host immune gene and transient expression of a T3 effector increase P. syringae growth in agromonas assays. We show that we can rapidly achieve structure-function analysis of immune components and test the function of immune hydrolases. The agromonas method is easy, fast and robust for routine disease assays with various Pseudomonas strains without transforming plants or bacteria. The agromonas assay offers reliable opportunity for further comprehensive analysis of plant immunity.One sentence summaryAgromonas is a rapid and robust disease assay to monitor Pseudomonas syringae growth in agroinfiltrated leaves expressing immune components and their suppressors.

scholarly journals Fasciclin-like arabinogalactan gene family in Nicotiana benthamiana: genome-wide identification, classification and expression in response to pathogens

2020 ◽  
Author(s):  
Xinyang Wu ◽  
Yuchao Lai ◽  
Lanqing Lv ◽  
Mengfei Ji ◽  
Kelei Han ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Nicotiana Benthamiana ◽  
Plant Pathogens ◽  
Plant Immunity ◽  
Arabinogalactan Proteins ◽  
Cis Acting ◽  
Genome Wide ◽  
A Genome ◽  
Polymerase Chain ◽  
Plant Pathogen Interactions

Abstract Background: Nicotiana benthamiana is widely used as a model plant to study plant-pathogen interactions. Fasciclin-like arabinogalactan proteins (FLAs), a subclass of arabinogalactan proteins (AGPs), participate in mediating plant growth, development and response to abiotic stress. However, the members of FLAs in N. benthamiana and their response to plant pathogens are unknown.Results: 38 NbFLAs were identified from a genome-wide study. NbFLAs could be divided into four subclasses, and their gene structure and motif composition were conserved in each subclass. NbFLAs may be regulated by cis-acting elements such as STRE and MBS, and could possibly be the targets of transcription factors like C2H2. Quantitative real time polymerase chain reaction (RT-qPCR) results showed that selected NbFLAs were differentially expressed in different tissues. All of the selected NbFLAs were significantly downregulated following infection by turnip mosaic virus (TuMV) and most of them also by Pseudomonas syringae pv tomato (Pst) strain DC3000 (Pst DC3000), suggesting possible roles in response to pathogenic infection.Conclusions: This study systematically identified FLAs in N. benthamiana, and indicates their potential roles in response to biotic stress. The identification of NbFLAs will facilitate further studies of their role in plant immunity in N. benthamiana.

scholarly journals Fasciclin-like arabinogalactan gene family in Nicotiana benthamiana: genome-wide identification, classification and expression in response to pathogens

2020 ◽  
Author(s):  
Xinyang Wu ◽  
Yuchao Lai ◽  
Lanqing Lv ◽  
Mengfei Ji ◽  
Kelei Han ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Nicotiana Benthamiana ◽  
Plant Pathogens ◽  
Plant Immunity ◽  
Arabinogalactan Proteins ◽  
Cis Acting ◽  
Genome Wide ◽  
A Genome ◽  
Polymerase Chain ◽  
Plant Pathogen Interactions

Abstract Background: Nicotiana benthamiana is widely used as a model plant to study plant-pathogen interactions. Fasciclin-like arabinogalactan proteins (FLAs), a subclass of arabinogalactan proteins (AGPs), participate in mediating plant growth, development and response to abiotic stress. However, the members of FLAs in N. benthamiana and their response to plant pathogens are unknown.Results: 38 NbFLAs were identified from a genome-wide study. NbFLAs could be divided into four subclasses, and their gene structure and motif composition were conserved in each subclass. NbFLAs may be regulated by cis-acting elements such as STRE and MBS, and may be the targets of transcription factors like C2H2. Quantitative real time polymerase chain reaction (RT-qPCR) results showed that selected NbFLAs were differentially expressed in different tissues. All of the selected NbFLAs were significantly downregulated following infection by turnip mosaic virus (TuMV) and most of them also by Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000), suggesting possible roles in response to pathogenic infection.Conclusions: This study systematically identified FLAs in N. benthamiana, and indicates their potential roles in response to biotic stress. The identification of NbFLAs will facilitate further studies of their role in plant immunity in N. benthamiana.

scholarly journals The Phytophthora sojae Avr1d Gene Encodes an RxLR-dEER Effector with Presence and Absence Polymorphisms Among Pathogen Strains

2013 ◽  
Vol 26 (8) ◽  
pp. 958-968 ◽  
Author(s):  
Weixiao Yin ◽  
Suomeng Dong ◽  
Luchong Zhai ◽  
Yachun Lin ◽  
Xiaobo Zheng ◽  
...  
Keyword(s):  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Plant Immunity ◽  
Phytophthora Sojae ◽  
Transcriptional Analysis ◽  
Mapping Data ◽  
Rxlr Effector ◽  
Effector Triggered Immunity ◽  
Oomycete Pathogen ◽  
Avr Genes

Soybean root and stem rot is caused by the oomycete pathogen Phytophthora sojae. The interaction between P. sojae and soybean fits the “gene-for-gene” hypothesis. Although more than 10 P. sojae avirulence (Avr) effectors have been genetically identified, nearly half of genetically defined avr genes have been cloned. In a previous bioinformatic and global transcriptional analysis, we identified a P. sojae RxLR effector, Avr1d, which was 125 amino acids in length. Mapping data demonstrated that Avr1d presence or absence in the genome was co-segregated with the Avr1d avirulence phenotype in F2 populations. Transient expression of the Avr1d gene using co-bombardment in soybean isogenic lines revealed that this gene triggered a hypersensitive response (HR) in the presence of Rps1d. Sequencing of Avr1d genes in different P. sojae strains revealed two Avr1d alleles. Although polymorphic, the two Avr1d alleles could trigger Rps1d-mediated HR. P. sojae strains carrying either of the alleles were avirulent on Rps1d soybean lines. Avr1d was upregulated during the germinating cyst and early infection stages. Furthermore, transient expression of Avr1d in Nicotiana benthamiana suppressed BAX-induced cell death and enhanced P. capsici infection. Avr1d also suppressed effector-triggered immunity induction by associating with Avr1b and Rps1b, suggestive of a role in suppressing plant immunity.

scholarly journals Genome-wide identification and analysis of Catharanthus roseus RLK1-like kinases in Nicotiana benthamiana

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shaofei Rao ◽  
Xinyang Wu ◽  
Hongying Zheng ◽  
Yuwen Lu ◽  
Jiejun Peng ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Catharanthus Roseus ◽  
Nicotiana Benthamiana ◽  
Expression Profiles ◽  
Plant Immunity ◽  
Expression Patterns ◽  
Gene Expression Profiles ◽  
Phylogenetic Groups ◽  
Specific Expression ◽  
Cis Acting

Abstract Background The Catharanthus roseus RLK1-like kinase (CrRLK1L) is a subfamily of the RLK gene family, and members are sensors of cell wall integrity and regulators of cell polarity growth. Recent studies have also shown that members of this subfamily are involved in plant immunity. Nicotiana benthamiana is a model plant widely used in the study of plant-pathogen interactions. However, the members of the NbCrRLK1L subfamily and their response to pathogens have not been reported. Results In this study, a total of 31 CrRLK1L members were identified in the N. benthamiana genome, and these can be divided into 6 phylogenetic groups (I-VI). The members in each group have similar exon-intron structures and conserved motifs. NbCrRLK1Ls were predicted to be regulated by cis-acting elements such as STRE, TCA, ABRE, etc., and to be the target of transcription factors such as Dof and MYB. The expression profiles of the 16 selected NbCrRLK1Ls were determined by quantitative PCR. Most NbCrRLK1Ls were highly expressed in leaves but there were different and diverse expression patterns in other tissues. Inoculation with the bacterium Pseudomonas syringae or with Turnip mosaic virus significantly altered the transcript levels of the tested genes, suggesting that NbCrRLK1Ls may be involved in the response to pathogens. Conclusions This study systematically identified the CrRLK1L members in N. benthamiana, and analyzed their tissue-specific expression and gene expression profiles in response to different pathogens and two pathogens associated molecular patterns (PAMPs). This research lays the foundation for exploring the function of NbCrRLK1Ls in plant-microbe interactions.

scholarly journals Structure-function analysis of ZAR1 immune receptor reveals key molecular interactions for activity

2019 ◽  
Author(s):  
Maël Baudin ◽  
Karl J. Schreiber ◽  
Eliza C. Martin ◽  
Andrei J. Petrescu ◽  
Jennifer D. Lewis
Keyword(s):  
Biological Sciences ◽  
Pseudomonas Syringae ◽  
Function Analysis ◽  
Plant Immunity ◽  
Expression System ◽  
Effector Proteins ◽  
Intramolecular Interactions ◽  
Immune Receptor ◽  
Romanian Academy ◽  
Transient System

ABSTRACTNLR (Nucleotide-binding [NB] Leucine-rich repeat [LRR] Receptor) proteins are critical for inducing immune responses in response to pathogen proteins, and must be tightly regulated to prevent spurious activation in the absence of a pathogen. The ZAR1 NLR recognizes diverse effector proteins from Pseudomonas syringae, including HopZ1a, and Xanthomonas species. Receptor-like cytoplasmic kinases (RLCKs) such as ZED1, interact with ZAR1 and provide specificity for different effector proteins, such as HopZ1a. We previously developed a transient expression system in Nicotiana benthamiana, that allowed us to demonstrate ZAR1 function is conserved from the Brassicaceae to the Solanaceae. Here, we combined structural modeling of ZAR1, with molecular and functional assays in our transient system, to show that multiple intramolecular and intermolecular interactions regulate ZAR1 activity. We identified new determinants required for the formation of the ZARCC dimer and its activity. Lastly, we characterized new intramolecular interactions between ZAR1 subdomains that participate in keeping ZAR1 immune complexes inactive. This work identifies molecular constraints on immune receptor function and activation.One sentence-summaryStructure-informed analyses reveal multiple finely-tuned intramolecular interactions that regulate the activity of the immune receptor ZAR1.FundingResearch on plant immunity in the Lewis laboratory was supported by the USDA ARS 2030-21000-046-00D and 2030-21000-050-00D (JDL), and the NSF Directorate for Biological Sciences IOS-1557661 (JDL). ECM and AJP acknowledge financial support from UEFISCDI grant PN-III-ID-PCE-2016-0650 and the Romanian Academy programs 1 & 2 of IBAR.

scholarly journals The Cytospora chrysosperma Virulence Effector CcCAP1 Mainly Localizes to the Plant Nucleus To Suppress Plant Immune Responses

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Zhu Han ◽  
Dianguang Xiong ◽  
Zhiye Xu ◽  
Tingli Liu ◽  
Chengming Tian
Keyword(s):  
Cell Death ◽  
Transient Expression ◽  
Nuclear Localization ◽  
Nicotiana Benthamiana ◽  
Plant Immunity ◽  
Host Cells ◽  
Inhibiting Activity ◽  
Content Type ◽  
Cytospora Chrysosperma ◽  
Plant Nucleus

ABSTRACT Canker disease is caused by the fungus Cytospora chrysosperma and damages a wide range of woody plants, causing major losses to crops and native plants. Plant pathogens secrete virulence-related effectors into host cells during infection to regulate plant immunity and promote colonization. However, the functions of C. chrysosperma effectors remain largely unknown. In this study, we used Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana and confocal microscopy to investigate the immunoregulation roles and subcellular localization of CcCAP1, a virulence-related effector identified in C. chrysosperma. CcCAP1 was significantly induced in the early stages of infection and contains cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily domain with four cysteines. CcCAP1 suppressed the programmed cell death triggered by Bcl-2-associated X protein (BAX) and the elicitin infestin1 (INF1) in transient expression assays with Nicotiana benthamiana. The CAP superfamily domain was sufficient for its cell death-inhibiting activity and three of the four cysteines in the CAP superfamily domain were indispensable for its activity. Pathogen challenge assays in N. benthamiana demonstrated that transient expression of CcCAP1 promoted Botrytis cinerea infection and restricted reactive oxygen species accumulation, callose deposition, and defense-related gene expression. In addition, expression of green fluorescent protein-labeled CcCAP1 in N. benthamiana showed that it localized to both the plant nucleus and the cytoplasm, but the nuclear localization was essential for its full immune inhibiting activity. These results suggest that this virulence-related effector of C. chrysosperma modulates plant immunity and functions mainly via its nuclear localization and the CAP domain. IMPORTANCE The data presented in this study provide a key resource for understanding the biology and molecular basis of necrotrophic pathogen responses to Nicotiana benthamiana resistance utilizing effector proteins, and CcCAP1 may be used in future studies to understand effector-triggered susceptibility processes in the Cytospora chrysosperma-poplar interaction system.

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

scholarly journals Using body temperature and variables commonly available in the EHR to predict acute infection: a proof-of-concept study showing improved pretest probability estimates for acute COVID-19 infection among discharged emergency department patients

Diagnosis ◽  
2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Carl T. Berdahl ◽  
An T. Nguyen ◽  
Marcio A. Diniz ◽  
Andrew J. Henreid ◽  
Teryl K. Nuckols ◽  
...  
Keyword(s):  
Emergency Department ◽  
Body Temperature ◽  
Acute Infection ◽  
Time Of Day ◽  
Pretest Probability ◽  
Absolute Difference ◽  
Proof Of Concept ◽  
Easy Method ◽  
Concept Study ◽  
Probability Estimates

Abstract Objectives Obtaining body temperature is a quick and easy method to screen for acute infection such as COVID-19. Currently, the predictive value of body temperature for acute infection is inhibited by failure to account for other readily available variables that affect temperature values. In this proof-of-concept study, we sought to improve COVID-19 pretest probability estimation by incorporating covariates known to be associated with body temperature, including patient age, sex, comorbidities, month, and time of day. Methods For patients discharged from an academic hospital emergency department after testing for COVID-19 in March and April of 2020, we abstracted clinical data. We reviewed physician documentation to retrospectively generate estimates of pretest probability for COVID-19. Using patients’ COVID-19 PCR test results as a gold standard, we compared AUCs of logistic regression models predicting COVID-19 positivity that used: (1) body temperature alone; (2) body temperature and pretest probability; (3) body temperature, pretest probability, and body temperature-relevant covariates. Calibration plots and bootstrap validation were used to assess predictive performance for model #3. Results Data from 117 patients were included. The models’ AUCs were: (1) 0.69 (2) 0.72, and (3) 0.76, respectively. The absolute difference in AUC was 0.029 (95% CI −0.057 to 0.114, p=0.25) between model 2 and 1 and 0.038 (95% CI −0.021 to 0.097, p=0.10) between model 3 and 2. Conclusions By incorporating covariates known to affect body temperature, we demonstrated improved pretest probability estimates of acute COVID-19 infection. Future work should be undertaken to further develop and validate our model in a larger, multi-institutional sample.

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