Reactive Oxygen Species (ROS) Measurement in Arabidopsis Guard Cells in Response to Biotic and Abiotic Stresses.

One of the major plant stress level indicators is reactive oxygen species (ROS). They have been known to play a central role in regulating plant responses to various environmental stresses. This book chapter specifically covers abiotic stress induced by a drought hormone abscisic acid and biotic stress induced by Pseudomonas syringe DC3000 on single cell-type guard cells. We describe in detail the measurement of ROS production starting from sample preparation to data analysis by fluorescence intensity acquisition using ImageJ software. We discussed the problems faced while performing the experiment and addressed how to overcome them by providing specific guidelines to ensure high quality repeatable data.

Combat phytopathogenic bacteria employing Argirium-SUNCs: limits and perspectives.

Bacterial plant diseases are difficult to control as the durability of deployed control measures is thwarted by continuous and rapid changing of bacterial populations. Although application of copper compounds to plants is the most widespread and inexpensive control measure, it is often partially efficacious for the frequent appearance of copper-resistant bacterial strains and it is raising concerns for the harmful effects of copper on environment and human health. Consequently, European Community included copper compounds in the list of substances candidates for substitution. Nanotechnologies and the application of nanoparticles seem to respond to the need to find new very effective and durable measures. We believe that Argirium-SUNCs®, silver ultra nanoclusters with an average size of 1.79 nm and characterized by rare oxidative states (Ag2+/3+), represent a valid candidate as a nano-bactericide in the control of plant bacterial diseases. Respect to the many silver nanoparticles described in the literature, Argirium-SUNCs have many strengths due to the reproducibility of the synthesis method, the purity and the stability of the preparation, the very strong (less than 1 ppm) antimicrobial, and anti-biofilm activities. In this mini-review, we provide information on this nanomaterial and on the possible application in agriculture. KEY POINTS: • Argirium-SUNCs have strong antimicrobial activities against phytopathogenic bacteria. • Argirium-SUNCs are a possible plant protection product. • Argirium-SUNCs protect tomato plants against bacterial speck disease.

Rice small secreted peptide, OsRALF26, recognized by FERONIA-like receptor 1 induces immunity in rice and Arabidopsis.

Rapid alkalinization factors (RALFs), belonging to a family of small secreted peptides, have been considered as important signaling molecules in diverse biological processes, including immunity. Current studies on RALF-modulated immunity mainly focus on Arabidopsis, but little is reported in crop plants. The rice immune receptor XA21 confers immunity to the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae (Xoo). Here, we pursued functional characterization of rice RALF26 (OsRALF26) up-regulated by Xoo during XA21-mediated immune response. When applied exogenously as a recombinant peptide, OsRALF26 induced a series of immune responses, including pathogenesis-related genes (PRs) induction, reactive oxygen species (ROS) production, and callose deposition in rice and/or Arabidopsis. Transgenic rice and Arabidopsis overexpressing OsRALF26 exhibited significantly enhanced resistance to Xoo and Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), respectively. In yeast two-hybrid, pull-down assays, and co-immunoprecipitation analyses, rice FER-like receptor 1 (OsFLR1) was identified as a receptor of OsRALF26. Transient expression of OsFLR1 in Nicotiana benthamiana leaves displayed significantly increased ROS production and callose deposition after OsRALF26 treatment. Together, we propose that OsRALF26 induced by Xoo in an XA21-dependent manner is perceived by OsFLR1 and may play a novel role in the enforcement of XA21-mediated immunity.

Voltage-dependent anion channel 3 (VDAC3) mediates P. syringae induced ABA-SA signaling crosstalk in Arabidopsis thaliana.

Pathogen severely affects plant mitochondrial processes including respiration, however, the roles and mechanism of mitochondrial protein during the immune response remain largely unexplored. The interplay of plant hormone signaling during defense is an outcome of plant pathogen interaction. We recently discovered that the Arabidopsis calcineurin B-like interacting protein kinase 9 (AtCIPK9) interacts with the voltage-dependent anion channel 3 (AtVDAC3) and inhibits MV-induced oxidative damage. Here we report the characterization of AtVDAC3 in an antagonistic interaction pathway between abscisic acid (ABA) and salicylic acid (SA) signaling in Pseudomonas syringae -Arabidopsis interaction. In this study, we observed that mutants of AtVDAC3 were highly susceptible to Pseudomonas syringae infection as compared to the wild type (WT) Arabidopsis plants. Transcripts of VDAC3 and CIPK9 were inducible upon ABA application. Following pathogen exposure, expression analyses of ABA and SA biosynthesis genes indicated that the function of VDAC3 is required for isochorisimate synthase 1 (ICS1) expression but not for Nine-cis-epoxycaotenoid dioxygenase 3 (NCED3) expression. Despite the fact that vdac3 mutants had increased NCED3 expression in response to pathogen challenge, transcripts of ABA sensitive genes such as AtRD22 and AtRAB18 were downregulated even after exogenous ABA application. VDAC3 is required for ABA responsive genes expression upon exogenous ABA application. We also found that Pseudomonas syringae-induced SA signaling is downregulated in vdac3 mutants since overexpression of VDAC3 resulted in hyperaccumulation of Pathogenesis related gene1 (PR1) transcript. Interestingly, ABA application prior to P. syringae inoculation resulted in the upregulation of ABA responsive genes like Responsive to ABA18 (RAB18) and Responsive to dehydration 22 (RD22). Intriguingly, in the absence of AtVDAC3, Pst challenge can dramatically increase ABA-induced RD22 and RAB18 expression. Altogether our results reveal a novel Pathogen-SA-ABA interaction pathway in plants. Our findings show that ABA plays a significant role in modifying plant-pathogen interactions, owing to cross-talk with the biotic stress signaling pathways of ABA and SA.

The Pseudomonas syringae pv. tomato DC3000 effector HopD1 interferes with cellular dynamics associated with the function of the plant immune protein AtNHR2B.

The plant pathogenic bacterium Pseudomonas syringae pv tomato DC3000 (Pst DC3000) causes disease in tomato, in the model plant Arabidopsis thaliana, and conditionally in Nicotiana benthamiana. The pathogenicity of Pst DC3000 is mostly due to bacterial virulence proteins, known as effectors, that are translocated into the plant cytoplasm through the type III secretion system (T3SS). Bacterial type III secreted effectors (T3SEs) target plants physiological processes and suppress defense responses to enable and support bacterial proliferation. The Pst DC3000 T3SE HopD1 interferes with plant defense responses by targeting the transcription factor NTL9. This work shows that HopD1 also targets the immune protein AtNHR2B (Arabidopsis thaliana nonhost resistance 2B), a protein that localizes to dynamic vesicles of the plant endomembrane system. Live-cell imaging of Nicotiana benthamiana plants transiently co-expressing HopD1 fused to the epitope haemagglutinin (HopD1-HA) with AtNHR2B fused to the red fluorescent protein (AtNHR2B-RFP), revealed that HopD1-HA interferes with the abundance and cellular dynamics of AtNHR2B-RFP-containing vesicles. The results from this study shed light into an additional function of HopD1 while contributing to understanding how T3SEs also target vesicle trafficking-mediated processes in plants.

Heritable induced resistance in Arabidopsis thaliana: Tips and tools to improve effect size and reproducibility.

Over a decade ago, three independent studies reported that pathogen- and herbivore-exposed Arabidopsis thaliana produces primed progeny with increased resistance. Since then, heritable induced resistance (h-IR) has been reported across numerous plant-biotic interactions, revealing a regulatory function of DNA (de)methylation dynamics. However, the identity of the epi-alleles controlling h-IR and the mechanisms by which they prime defense genes remain unknown, while the evolutionary significance of the response requires confirmation. Progress has been hampered by the relatively high variability, low effect size, and sometimes poor reproducibility of h-IR, as is exemplified by a recent study that failed to reproduce h-IR in A. thaliana by Pseudomonas syringae pv. tomato (Pst). This study aimed to improve h-IR effect size and reproducibility in the A. thaliana-Pst interaction. We show that recurrent Pst inoculations of seedlings result in stronger h-IR than repeated inoculations of older plants and that disease-related growth repression in the parents is a reliable marker for h-IR effect size in F1 progeny. Furthermore, RT-qPCR-based expression profiling of genes controlling DNA methylation maintenance revealed that the elicitation of strong h-IR upon seedling inoculations is marked by reduced expression of the chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1) gene, which is maintained in the apical meristem and transmitted to F1 progeny. Two additional genes, MET1 and CHROMOMETHYLASE3 (CMT3), displayed similar transcriptional repression in progeny from seedling-inoculated plants. Thus, reduced expression of DDM1, MET1, and CMT3 can serve as a marker of robust h-IR in F1 progeny. Our report offers valuable information and markers to improve the effect size and reproducibility of h-IR in the A. thaliana-Pst model interaction.

Race-specific genotypes of Pseudomonas syringae pv. tomato are defined by the presence of mobile DNA elements within the genome.

Pseudomonas syringae pv. tomato is the causal agent of bacterial speck of tomato, an important disease that results in severe crop production losses worldwide. Currently, two races within phylogroup 01a (PG01a) are described for this pathogen. Race 0 strains have avirulence genes for the expression of type III system-associated effectors AvrPto1 and AvrPtoB, that are recognized and targeted by the effector-triggered immunity in tomato cultivars having the pto race-specific resistance gene. Race 1 strains instead lack the avrPto1 and avrPtoB genes and are therefore capable to aggressively attack all tomato cultivars. Here, we have performed the complete genome sequencing and the analysis of P. syringae pv. tomato strain DAPP-PG 215, which was described as a race 0 strain in 1996. Our analysis revealed that its genome comprises a 6.2 Mb circular chromosome and two plasmids (107 kb and 81 kb). The results indicate that the strain is phylogenetically closely related to strains Max13, K40, T1 and NYS-T1, all known race 1 strains. The chromosome of DAPP-PG 215 encodes race 1-associated genes like avrA and hopW1 and lacks race 0-associated genes like hopN1, giving it a race 1 genetic background. However, the genome harbors a complete ortholog of avrPto1, which allows the strain to display a race 0 phenotype. Comparative genomics with several PG01a genomes revealed that mobile DNA elements are rather involved in the evolution of the two different races.

Silver nanoclusters with Ag2+/3+ oxidative states are a new highly effective tool against phytopathogenic bacteria.

The main measure worldwide adopted to manage plant bacterial diseases is based on the application of copper compounds, which are often partially efficacious for the frequent appearance of copper-resistant bacterial strains and have raised concerns for their toxicity to the environment and humans. Therefore, there is an increasing need to develop new environmentally friendly, efficient, and reliable strategies for controlling plant bacterial diseases, and among them, the use of nanoparticles seems promising. The present study aimed to evaluate the feasibility of protecting plants against attacks of gram-negative and gram-positive phytopathogenic bacteria by using electrochemically synthesized silver ultra nanoclusters (ARGIRIUM­SUNCs®) with an average size of 1.79 nm and characterized by rare oxidative states (Ag2+/3+). ARGIRIUM­SUNCs strongly inhibited the in vitro growth (effective concentration, EC50, less than 1 ppm) and biofilm formation of Pseudomonas syringae pv. tomato and of quarantine bacteria Xanthomonas vesicatoria, Xylella fastidiosa subsp. pauca, and Clavibacter michiganensis subsp. michiganensis. In addition, treatments with ARGIRIUM­SUNCs also provoked the eradication of biofilm for P. syringae pv. tomato, X. vesicatoria, and C. michiganensis subsp. michiganensis. Treatment of tomato plants via root absorption with ARGIRIUM­SUNCs (10 ppm) is not phytotoxic and protected (80%) the plants against P. syringae pv. tomato attacks. ARGIRIUM­SUNCs at low doses induced hormetic effects on P. syringae pv. tomato, X. vesicatoria, and C. michiganensis subsp. michiganensis as well as on tomato root growth. The use of ARGIRIUM­SUNCs in protecting plants against phytopathogenic bacteria is a possible alternative control measure. KEY POINTS: • ARGIRIUM­SUNC has strong antimicrobial activities against phytopathogenic bacteria; • ARGIRIUM­SUNC inhibits biofilm formation at low doses; • ARGIRIUM­SUNC protects tomato plants against bacterial speck disease.

Loss-of-function mutations in WRKY22 and WRKY25 impair stomatal-mediated immunity and PTI and ETI responses against Pseudomonas syringae pv. tomato.

Plants defend themselves against pathogens using a two-layered immune system. The first response, pattern-triggered immunity (PTI), is activated upon recognition of microbe-associated molecular patterns (MAMPs). Virulent bacteria such as Pseudomonas syringae pv. tomato (Pst), deliver effector proteins into the plant cell to promote susceptibility. However, some plants possess resistance (R) proteins that recognize specific effectors leading to the activation of the second response, effector-triggered immunity (ETI). Resistant tomatoes such as Río Grande-PtoR recognize two Pst effectors (AvrPto and AvrPtoB) through the host Pto/Prf complex and activate ETI. We previously showed that the transcription factors (TF) WRKY22 and WRKY25 are positive regulators of plant immunity against bacterial and potentially non-bacterial pathogens in Nicotiana benthamiana. Here, the CRISPR-Cas9 technique was used to develop three knockout tomato lines for either one or both TFs. The single and double mutants were all compromised in Pto/Prf-mediated ETI and had a weaker PTI response. The stomata apertures in all of the mutant lines did not respond to darkness or challenge with Pst DC3000. The WRKY22 and WRKY25 proteins both localize in the nucleus, but we found no evidence of a physical interaction between them. The WRKY22 TF was found to be involved in the transcriptional regulation of WRKY25, supporting the idea that they are not functionally redundant. Together, our results indicate that both WRKY TFs play a role in modulating stomata and are positive regulators of plant immunity in tomato.

In planta interactions of a novel bacteriophage against Pseudomonas syringae pv. tomato.

The biology and biotechnology of bacteriophages have been extensively studied in recent years to explore new and environmentally friendly methods of controlling phytopathogenic bacteria. Pseudomonas syringae pv. tomato (Pst) is responsible for bacterial speck disease in tomato plants, leading to decreased yield. Disease management strategies rely on the use of copper-based pesticides. The biological control of Pst with the use of bacteriophages could be an alternative environmentally friendly approach to diminish the detrimental effects of Pst in tomato cultivations. The lytic efficacy of bacteriophages can be used in biocontrol-based disease management strategies. Here, we report the isolation and complete characterization of a bacteriophage, named Medea1, which was also tested in planta against Pst, under greenhouse conditions. The application of Medea1 as a root drenching inoculum or foliar spraying reduced 2.5- and fourfold on average, respectively, Pst symptoms in tomato plants, compared to a control group. In addition, it was observed that defense-related genes PR1b and Pin2 were upregulated in the phage-treated plants. Our research explores a new genus of Pseudomonas phages and explores its biocontrol potential against Pst, by utilizing its lytic nature and ability to trigger the immune response of plants. KEY POINTS: • Medea1 is a newly reported bacteriophage against Pseudomonas syringae pv. tomato having genomic similarities with the phiPSA1 bacteriophage • Two application strategies were reported, one by root drenching the plants with a phage-based solution and one by foliar spraying, showing up to 60- and 6-fold reduction of Pst population and disease severity in some cases, respectively, compared to control • Bacteriophage Medea1 induced the expression of the plant defense-related genes Pin2 and PR1b.

The enzymatic hydrolysate of fucoidan from Sargassum hemiphyllum triggers immunity in plants.

Fucoidans are polysaccharides that consist predominantly of sulfated L-fucoses, from which, fucoidan oligosaccharides (FOSs) are prepared through different methods. Fucoidan has versatile physiological activities, like antiviral functions against SARS CoV-2 and bioactivitiy in enhancing immune responses. Although fucoidan or FOS has been widely used in mammals as functional foods and new drugs, its application in plants is still very limited. Moreover, whether fucoidan or its derived hydrolytic products can trigger immune responses in plants remained unknown. In this work, we demonstrate that the fucoidan enzymatic hydrolysate (FEH) prepared from Sargassum hemiphyllum triggers various immune responses, such as ROS production, MAPK activation, gene expression reprogramming, callose deposition, stomatal closure, and plant resistance to the bacterial strain Pseudomonas syringae pv. tomato (Pst) DC3000. Notably, FEH did not induce Arabidopsis root growth inhibition at the concentration used for triggering other immune responses. Our work suggests that EHF can potentially be used as a non-microbial elicitor in agricultural practices to protect plants from pathogen infection.

Rootstock increases the physiological defence of tomato plants against Pseudomonas syringae pv. tomato infection.

Climate change has intensified the infection of tomato plants by pathogens such as Pseudomonas syringae pv. tomato (Pst). Rootstocks may increase plant tolerance to leaf phytopathogens. The aim of this study was to evaluate the effects of the tolerant Poncho Negro (R) tomato rootstock on physiological defence and the role of hydrogen sulfide (H2S) in susceptible Limachino (L) tomato plant responses to Pst attack. Ungrafted (L), self-grafted (L/L), and grafted (L/R) plants were infected with Pst. Rootstock increased the concentration of antioxidant compounds including ascorbate in the scion. Tolerant rootstock induced an increase of H2S in the scion, which correlated with enhanced expression of the SlAPX2 gene. A high accumulation of salicylic acid was observed in Pst-inoculated grafted L/L and L/R plants, but this was higher in L/R plants. The increase of H2S during Pst infection was associated with a reduction of ethylene in L/R plants. Our study indicates that the Poncho Negro rootstock reduced the symptoms of bacterial speck disease in the Limachino tomato plants, conferring tolerance to Pst infection. This study provides new knowledge about the impact of rootstock in the defence of tomato plants against leaf pathogens that could be used in sustainable management of tomato cultivation.

Overlapping Local and Systemic Defense Induced by an Oomycete Fatty Acid MAMP and Brown Seaweed Extract in Tomato.

Eicosapolyenoic fatty acids are integral components of oomycete pathogens that can act as microbe-associated molecular patterns to induce disease resistance in plants. Defense-inducing eicosapolyenoic fatty acids include arachidonic acid (AA) and eicosapentaenoic acid and are strong elicitors in solanaceous plants, with bioactivity in other plant families. Similarly, extracts of a brown seaweed, Ascophyllum nodosum, used in sustainable agriculture as a biostimulant of plant growth, may also induce disease resistance. A. nodosum, similar to other macroalgae, is rich in eicosapolyenoic fatty acids, which comprise as much as 25% of total fatty acid composition. We investigated the response of roots and leaves from AA or a commercial A. nodosum extract (ANE) on root-treated tomatoes via RNA sequencing, phytohormone profiling, and disease assays. AA and ANE significantly altered transcriptional profiles relative to control plants, inducing numerous defense-related genes with both substantial overlap and differences in gene expression patterns. Root treatment with AA and, to a lesser extent, ANE also altered both salicylic acid and jasmonic acid levels while inducing local and systemic resistance to oomycete and bacterial pathogen challenge. Thus, our study highlights overlap in both local and systemic defense induced by AA and ANE, with potential for inducing broad-spectrum resistance against pathogens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Xanthomonas natriuretic peptide is recognized by the Arabidopsis natriuretic peptide receptor 1 and through this interaction triggers similar plant responses to its plant counterpart.

Plant natriuretic peptides (PNPs) are hormone peptides that participate in the regulation of ions and water homeostasis in plants. Xanthomonas citri subsp. citri (Xcc) the causal agent of citrus canker disease also possesses a PNP-like peptide (XacPNP). This peptide, similarly to AtPNP-A, the most studied PNP from Arabidopsis thaliana, causes stomatal aperture and enhances photosynthetic efficiency in plant leaves. Thus, the function that has been attributed to XacPNP is to contribute to maintain photosynthetic efficiency and water homeostasis in plant tissue during the infection process, to create favorable conditions for biotrophic pathogens survival. A PNP receptor (AtPNP-R1) for AtPNP-A has been identified and the AtPNP-A activity in regulation of water homeostasis has been observed to depend on the presence of AtPNP-R1. Here, we demonstrated that both AtPNP-A and XacPNP require the presence of AtPNP-R1 to induce plant stomatal aperture. Also, less necrotic tissue was found in infections with pathogens expressing XacPNP and this was dependent on the presence of AtPNP-R1, suggesting that XacPNP interacts with this receptor to exert its function. Finally, we confirmed that AtPNP-A and XacPNP interact with AtPNP-R1 in planta, which support the idea that XacPNP triggers similar plant responses to its plant counterpart.

RING-Type E3 Ubiquitin Ligases AtRDUF1 and AtRDUF2 Positively Regulate the Expression of PR1 Gene and Pattern-Triggered Immunity.

The importance of E3 ubiquitin ligases from different families for plant immune signaling has been confirmed. Plant RING-type E3 ubiquitin ligases are members of the E3 ligase superfamily and have been shown to play positive or negative roles during the regulation of various steps of plant immunity. Here, we present Arabidopsis RING-type E3 ubiquitin ligases AtRDUF1 and AtRDUF2 which act as positive regulators of flg22- and SA-mediated defense signaling. Expression of AtRDUF1 and AtRDUF2 is induced by pathogen-associated molecular patterns (PAMPs) and pathogens. The atrduf1 and atrduf2 mutants displayed weakened responses when triggered by PAMPs. Immune responses, including oxidative burst, mitogen-activated protein kinase (MAPK) activity, and transcriptional activation of marker genes, were attenuated in the atrduf1 and atrduf2 mutants. The suppressed activation of PTI responses also resulted in enhanced susceptibility to bacterial pathogens. Interestingly, atrduf1 and atrduf2 mutants showed defects in SA-mediated or pathogen-mediated PR1 expression; however, avirulent Pseudomonas syringae pv. tomato DC3000-induced cell death was unaffected. Our findings suggest that AtRDUF1 and AtRDUF2 are not just PTI-positive regulators but are also involved in SA-mediated PR1 gene expression, which is important for resistance to P. syringae.

Transcriptomic and Metabolomic Analysis of a Pseudomonas-Resistant versus a Susceptible Arabidopsis Accession.

Accessions of one plant species may show significantly different levels of susceptibility to stresses. The Arabidopsis thaliana accessions Col-0 and C24 differ significantly in their resistance to the pathogen Pseudomonas syringae pv. tomato (Pst). To help unravel the underlying mechanisms contributing to this naturally occurring variance in resistance to Pst, we analyzed changes in transcripts and compounds from primary and secondary metabolism of Col-0 and C24 at different time points after infection with Pst. Our results show that the differences in the resistance of Col-0 and C24 mainly involve mechanisms of salicylic-acid-dependent systemic acquired resistance, while responses of jasmonic-acid-dependent mechanisms are shared between the two accessions. In addition, arginine metabolism and differential activity of the biosynthesis pathways of aliphatic glucosinolates and indole glucosinolates may also contribute to the resistance. Thus, this study highlights the difference in the defense response strategies utilized by different genotypes.

Photosensitizer to the rescue: in planta and field application of photodynamic inactivation against plant pathogenic bacteria.

Control of plant pathogens using chemical and synthetic pesticides raises a major safety concern for humans and the environment. Despite the ongoing exploration of sustainable alternative methods, management practices for pathogens, especially bacteria, have remained almost unchanged over decades, whereby long-term uses of copper and antibiotics has led to widespread bacterial resistance in the field. Antimicrobial photodynamic inactivation (aPDI) of bacteria is emerging as an alternative strategy to combat resistant plant pathogens. aPDI utilizes light-sensitive molecules (photosensitizers) that upon illumination produce reactive oxygen species able to kill pathogens. Here we explore the potential of an anionic semisynthetic water-soluble derivative of chlorophyl (Sodium Magnesium Chlorophyllin: Mg-chl), as an antibacterial agent in planta, by simulating processes naturally occurring in the field. Mg-chl in combination with Na2EDTA (cell wall permeabilizing agent) was able to effectively inhibit Pseudomonas syringae pv. tomato DC3000 in vitro and in planta in both tomato and N. benthamiana. Notably, Mg-chl in combination with Na2EDTA and the common surfactant Morwet D-400 significantly reduced Xanthomonas hortorum pv. gardneri and Xanthomonas fragarie, respectively, in a commercial greenhouse trial against bacterial spot disease in tomato and in field experiments against angular leaf spot disease in strawberries.

Thiamine Is Required for Virulence and Survival of Pseudomonas syringae pv. tomato DC3000 on Tomatoes.

Pseudomonas syringae pv. tomato DC3000 (PstDC3000) is an important plant pathogen that infects tomatoes and Arabidopsis. Thiamine and its derivative thiamine pyrophosphate (TPP) are cofactors that play an important role in the growth and survival of many bacterial microorganisms. However, the role of thiamine-related genes has not been determined in PstDC3000. Hence, to investigate the role of TPP in growth, resistance to stresses, and virulence of PstDC3000, double and quadruple mutants of thiamine biosynthesis-related genes (thiD/E, thiS/G, and thiD/E/S/G deletion mutants) as well as a single mutant of a lipoprotein-related gene (apbE) were constructed. Our results showed that growth of the thiD/E, thiS/G, and thiD/E/S/G mutants in the mannitol-glutamate (MG) medium was significantly lower than that of the wild type (WT) and their growth could be restored to the WT level with the addition of exogenous thiamine, whereas mutation of the apbE gene did not affect its growth in vitro. While tolerance to acid, osmotic, and oxidative stresses for the double mutants was similar to the WT, tolerance to stresses for the apbE mutant was reduced as compared to the WT. In addition, all four mutants exhibited reduced virulence and growth in tomatoes. However, when the double and quadruple mutants were inoculated with exogenous thiamine, the virulence and growth rate of these mutants were restored to the WT level. These results indicated that the thiD/E, thiS/G, and thiD/E/S/G mutants exhibiting growth deficiency in planta are probably due to a lack of thiamine biosynthesis, thus reducing colonization in tomatoes. On the other hand, it is possible that the apbE mutant exhibited reduced stress tolerances, thus resulting in reduced colonization. Overall, our findings suggest that the thiamine biosynthetic (TBS) pathway plays an important role in the colonization and infection of PstDC3000. Therefore, the thiamine biosynthetic pathway could be used as the target to develop new control measures for a bacterial spot in tomatoes.

Cysteine Reactivity Profiling to Unveil Redox Regulation in Phytopathogens.

Reactivity-based chemical proteomics is a powerful technology based on the use of tagged chemicals that covalently react with surface-exposed residues on proteins in native proteomes. Reactivity profiling involves the purification, identification, and quantification of labeled peptides by LC-MS/MS. Here, we have detailed a protocol for reactivity profiling of Cys residues using iodoacetamide probes, displaying >1000 reactive Cys residues in the proteome of phytopathogen Pseudomonas syringae pv. tomato DC3000 (PtoDC3000). Comparative reactivity profiling of PtoDC3000 treated with or without hydrogen peroxide (H2O2) identified ~200 H2O2-sensitive Cys residues in antioxidant enzymes, metabolic enzymes, and transcription regulators. Interestingly, half of these H2O2-sensitive Cys residues are more reactive in response to H2O2 and several proteins have multiple Cys residues with opposite reactivities in response to H2O2 exposure.

Detection and Quantification of the Hypersensitive Response Cell Death in Arabidopsis thaliana.

In plants, the hypersensitive response (HR) is a programmed cell death modality that occurs upon recognition of harmful non-self. It occurs at the site of pathogen infection, thus preventing pathogens to live off plant tissue and proliferate. Shedding light on the molecular constituents underlying this process requires robust and quantitative methods that can determine whether plants lacking functional genes are defective in HR execution compared to wild-type controls. In this chapter, we provide two quantitative protocols in which we measure cell death from Arabidopsis thaliana leaves infected with avirulent HR-causing bacterial strains. Firstly, we use trypan blue staining to quantify the stained area of leaves upon bacterial infection using a personalized macro in the Image J (Fiji) software. Alternately, we incorporate an electrolyte leakage protocol in order to measure HR caused by different avirulent bacterial strains at different bacterial titers. We encourage users to perform a combination of both methods when assessing HR in different plant genotypes.