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.

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.

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.