Validation of RT-qPCR approaches to monitor<i>Pseudomonas syringae</i>gene expression during infection and exposure to pattern-triggered immunity

Smith, Amy M.; Lovelace, Amelia H.; Kvitko, Brian H.

doi:10.1101/183269

Cited by 4 publications

(8 citation statements)

References 45 publications

(68 reference statements)

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“…For example, the expression of rpoD at 6 hours post-inoculation was almost 200 times greater than in the inoculum, and 6 times greater than the expression at 168 hours. rpoD has been used in the past as a qPCR reference gene (Smith et al 2018); however, its expression, at least under certain conditions, is too variable for this purpose, as observed in our experiments. ftsZ showed the same trend of early increased expression and subsequent decrease in planta; however, the relative expression in the inoculum was more variable (Figure 7A and Supplementary Figure S8B).…”

Section: Bacterial Gene Expression Of Select Biological Processes During Long-term Adaptation Of a Non-pathogen Inside Plantsmentioning

confidence: 79%

Section: Resultsmentioning

confidence: 99%

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Phyllosphere-inhabiting endophytic bacteria feature a stationary phase-like lifestyle

2021

Preprint

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Plants are in constant association with a variety of microbes, and although much is known about how symbiotic and pathogenic microbes interact with plants, less is known about the population dynamics, adaptive traits, and transcriptional features of endophytic commensal microbes that live inside leaves. In this study, we evaluated the long-term population and transcriptional dynamics of two bacterial microbiota endophytes and compared them to those of a commensal-simulating non-pathogenic mutant of the bacterial pathogen Pseudomonas syringae. We found that population densities of all three endophytic phyllosphere bacteria remained static over a long period of time, which was caused by a continual equilibrium between bacterial multiplication and death, as evidenced by treatment of plants with antibiotics that only targeted dividing bacteria or by in planta visualization of bacteria carrying a fluorescent division reporter. Population stasis could not be explained by a lack of resources, as Arabidopsis leaves could support population densities up to 100 times higher than the normal microbiota populations, nor was population stasis reversed by significantly quenching PAMP-triggered immunity. Long-term temporal in planta transcriptomic analysis of these three bacterial endophytes revealed up-regulation of protein translation, the generation of energy, and the response to stress, and interestingly, for the microbiota strains, the longer the bacteria remained inside plants, the greater the up-regulation of some of these processes. Further transcriptomic analysis of in planta populations of commensal-simulating Pseudomonas syringae revealed a remarkable resemblance to those of in vitro bacteria in stationary phase, a metabolically active physiological state in which the production of secondary metabolites and stress responses are induced. This study provides novel insight into how endophytic bacteria survive and thrive inside plant leaves, and reshapes our current understanding of what it means to be part of the endophytic microbiota in the phyllosphere.

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Section: Bacterial Gene Expression Of Select Biological Processes During Long-term Adaptation Of a Non-pathogen Inside Plantsmentioning

confidence: 79%

Section: Resultsmentioning

confidence: 99%

Phyllosphere-inhabiting endophytic bacteria feature a stationary phase-like lifestyle

2021

Preprint

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“…RT-qPCR data were normalized to recA (PSPTO_4033) and rpoD (PSPTO_0537) used as reference genes. The stability of recA and rpoD expression in planta at 24 and 48 h after inoculation was confirmed as described by Smith et al (36), using a synthetic double-stranded DNA gBlock (Integrated DNA Technologies, Coralville, IA, USA) and PtoDC3000-specific 16S rRNA primers.…”

Section: Monitoring Bacterial Gene Expression In Plantamentioning

confidence: 99%

“…We assessed the stability of expression of several house-keeping genes in order to choose those suitable for use as reference genes for normalization of gene expression (36) and found that recA and rpoD were expressed at very similar levels at 24 and 48 hours after inoculation.…”

Section: Auxin Modulates Bacterial Virulence-related Gene Expression mentioning

confidence: 99%

“…The total RNA obtained was a mix of bacterial and plant RNAs. We used the concentration of PtoDC3000-specific 16S rRNA (PSPTO_r01) as an in-sample proxy for bacterial concentration within the mixed RNA samples as described recently by Smith et al (36). In this approach, the concentrations of bacterial RNA in the total RNA isolated from infected plant tissue were standardized using PtoDC3000-specific 16S ribosomal RNA primers.…”

Section: Monitoring Bacterial Gene Expression In Plantamentioning

confidence: 99%

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Dual role of auxin in regulating plant defense and bacterial virulence gene expression duringPseudomonas syringae PtoDC3000 pathogenesis

Djami-Tchatchou

Harrison

Harper

et al. 2019

Preprint

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ABSTRACTModification of host hormone biology is a common strategy used by plant pathogens to promote disease. For example, the bacterial pathogen Pseudomonas syringae strain PtoDC3000 produces the plant hormone auxin (Indole-3-acetic acid, or IAA) to promote PtoDC3000 growth in plant tissue. Previous studies suggest that auxin may promote PtoDC3000 pathogenesis through multiple mechanisms, including both suppression of salicylic acid (SA)-mediated host defenses and via an unknown mechanism that appears to be independent of SA. To test if host auxin signaling is important during pathogenesis, we took advantage of Arabidopsis thaliana lines impaired in either auxin signaling or perception. We found that disruption of auxin signaling in plants expressing an inducible dominant axr2-1 mutation resulted in decreased bacterial growth, demonstrating that host auxin signaling is required for normal susceptibility to PtoDC3000, and this phenotype was dependent on SA-mediated defenses. However, despite exhibiting decreased auxin perception, tir1 afb1 afb4 afb5 quadruple mutant plants lacking four of the six known auxin co-receptors supported increased levels of bacterial growth. This mutant also exhibited elevated IAA levels, suggesting that the increased IAA in these plants may promote PtoDC3000 growth independent of host auxin signaling, perhaps through a direct effect on the pathogen. In support of this, we found that IAA directly impacted the pathogen, by modulating expression of bacterial virulence genes, both in liquid culture and in planta. Thus, in addition to suppressing host defenses, IAA acts as a microbial signaling molecule that regulates bacterial virulence gene expression.

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Elicitors of plant defense induce the accumulation of amino acids that suppress both bacterial virulence and growth

Zhang

Tubergen

Agorsor

et al. 2022

Preprint

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Plant immunity relies on the perception of Microbe-Associate Molecular Patterns (MAMPs) from invading microbes to induce defense responses that suppress attempted infections. It has been proposed that MAMP-triggered immunity (MTI) suppresses bacterial infections by suppressing the onset of bacterial virulence. However, the mechanisms by which plants exert this action are poorly understood. Here we uncover that MAMP-perception in Arabidopsis induces the accumulation of free amino acids (AA) in a salicylic acid (SA) dependent manner. When co-infiltrated with Glutamine and Serine, two of the MAMP-induced highly accumulating amino acids, Pseudomonas syringae pv. tomato DC3000 expressed low levels of virulence genes and failed to produce robust infections in otherwise susceptible plants. When applied exogenously, Glutamine and Serine suppressed bacterial virulence and bacterial growth directly without the need for MAMP-perception and SA-signaling, bypassing MAMP-elicited defense. In addition, an increased level of endogenous Glutamine in the leaf apoplast of a gain-of-function mutant of Glutamine Dumper-1 rescued the partially compromised bacterial virulence and growth suppressing phenotype of the SA-induced deficient-2 (sid2) mutant. Our data suggest that MTI suppresses bacterial infections by the direct suppressing effect of accumulating amino acids on the onset of bacterial virulence.

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Validation of RT-qPCR approaches to monitorPseudomonas syringaegene expression during infection and exposure to pattern-triggered immunity

Cited by 4 publications

References 45 publications

Phyllosphere-inhabiting endophytic bacteria feature a stationary phase-like lifestyle

Phyllosphere-inhabiting endophytic bacteria feature a stationary phase-like lifestyle

Dual role of auxin in regulating plant defense and bacterial virulence gene expression duringPseudomonas syringae PtoDC3000 pathogenesis

Elicitors of plant defense induce the accumulation of amino acids that suppress both bacterial virulence and growth

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