Pattern-triggered immunity (PTI) can confer broad defense against diverse microbes and pathogens with disparate lifestyles through the detection of microbial extracellular signatures by surface-exposed pattern recognition receptors. However, unlike recognition of pathogen effectors by cytosolic resistance proteins, PTI is typically not associated with a host-cell programmed cell death response. Although host PTI signaling has been extensively studied, the mechanisms by which it restricts microbial colonization are poorly understood. We sought to gain insight into the mechanisms of PTI action by using bacterial transcriptomics analysis during exposure to PTI. Here, we describe a method for bacterial cell extraction from inoculated leaves that was used to analyze a time course of genome-wide transcriptional responses in the pathogen Pseudomonas syringae pv. tomato DC3000 during early naïve host infection and exposure to pre-induced PTI in Arabidopsis thaliana. Our analysis revealed early transcriptional regulation of important bacterial metabolic processes and host interaction pathways. We observed peak induction of P. syringae virulence genes at 3 h postinoculation and that exposure to PTI was associated with significant reductions in the expression of virulence genes. We also observed the induction of P. syringae sulfur starvation response genes such as sulfate and sulfonate importers only during exposure to PTI.

Pseudomonas syringae pv. tomato DC3000 is an important model plant pathogen, with a fully annotated genome and multiple compatible plant hosts. Very few studies have examined the regulation of DC3000 gene expression in vivo. We developed a quantitative reverse transcription-polymerase chain reaction assay to monitor transcriptional changes in DC3000 inoculated into Arabidopsis thaliana leaves during disease and exposure to pattern-triggered immunity (PTI). In our approach, bacterial RNA concentrations in total tissue RNA are standardized using P. syringae-specific 16S ribosomal RNA primers. We validated multiple stable reference genes for normalization in calculating the relative expression of genes of interest. We used empirically derived rates of amplification efficiency to calculate relative expression of key marker genes for virulence-associated regulation. We demonstrated that exposure to PTI alters DC3000 expression of type III secretion system, coronatine synthesis genes, and flagellar marker genes.

Effectors play a central role in determining the outcome of plant−pathogen interactions. As key virulence proteins, effectors are collectively indispensable for disease development. By understanding the virulence mechanisms of effectors, fundamental knowledge of microbial pathogenesis and disease resistance have been revealed. Effectors are also considered double-edged swords because some of them activate immunity in disease resistant plants after being recognized by specific immune receptors, which evolved to monitor pathogen presence or activity. Characterization of effector recognition by their cognate immune receptors and the downstream immune signaling pathways is instrumental in implementing resistance. Over the past decades, substantial research effort has focused on effector biology, especially concerning their interactions with virulence targets or immune receptors in plant cells. A foundation of this research is robust identification of the effector repertoire from a given pathogen, which depends heavily on bioinformatic prediction. In this review, we summarize methodologies that have been used for effector mining in various microbial pathogens which use different effector delivery mechanisms. We also discuss current limitations and provide perspectives on how recently developed analytic tools and technologies may facilitate effector identification and hence generation of a more complete vision of host−pathogen interactions. [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 .

Pseudomonas syringae pv. tomato DC3000 (DC3000) is an important model plant pathogen, with a fully annotated genome and multiple compatible plant hosts. Very few studies have examined the regulation of DC3000 gene expression in vivo. We developed a RT-qPCR assay to monitor transcriptional changes in DC3000 inoculated into Arabidopsis thaliana leaves during disease and exposure to pattern-triggered immunity (PTI). In our approach, bacterial RNA concentrations in total tissue RNA are standardized using P.syringae-specific16S ribosomal RNA primers. We validated multiple stable reference genes for normalization in calculating the relative expression of genes of interest. We used empirically derived rates of amplification efficiency to calculate relative expression of key marker genes for virulence-associated regulation. We demonstrated that exposure to PTI alters DC3000 expression of Type III secretion system, coronatine synthesis genes and flagellar marker genes.