Agromonas: a rapid disease assay for <i>Pseudomonas syringae</i> growth in agroinfiltrated leaves

Buscaill, Pierre; Sanguankiattichai, Nattapong; Lee, Yoon Joo; Kourelis, Jiorgos; Preston, Gail M.; Hoorn, Renier A. L. van der

doi:10.1111/tpj.15056

Cited by 21 publications

(8 citation statements)

References 59 publications

(71 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To test the possible requirement of Pic3 phosphatase activity in defense, we generated a construct carrying a variant, Pic3NN, with substitutions at the two conserved aspartic residues (D71N and D233N; Figure 2A ). Using the ‘agromonas’ approach (Buscaill et al, 2021), we transiently expressed Pic3, Pic3NN and a YFP control for 2 days in N. benthamiana leaves and then inoculated the plants with DC3000 ΔhopQ1-1, which lacks the T3SS effector gene hopQ1-1 that can be detected by the NLR protein Recognition of XopQ 1 (Roq1), thus making DC3000 ΔhopQ1-1 virulent in N. benthamiana; (Wei et al, 2007; Schultink et al, 2017). Compared to the YFP control, transient expression of wild-type Pic3 significantly increased the DC3000 ΔhopQ1-1 population in N. benthamiana, consistent with Pic3 playing a negative role in resistance to DC3000 ΔhopQ1-1 ( Figure 2B ).…”

Section: Resultsmentioning

confidence: 99%

Related PP2C phosphatases Pic3 and Pic12 negatively regulate immunity in tomato toPseudomonas syringae

Xia,

Zhang,

Smith

et al. 2024

Preprint

View full text Add to dashboard Cite

Type 2C protein phosphatases (PP2Cs) constitute a large family in most plant species but relatively few of them have been implicated in immunity. To identify and characterize PP2C phosphatases that affect tomato immunity, we used CRISPR/Cas9 to generate loss-of-function mutants for 11 PP2C genes whose expression is altered in response to immune elicitors or pathogens. We report that two closely-related tomato PP2C phosphatases, Pic3 and Pic12, are involved in regulating resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Loss-of-function mutations in Pic3 lead to enhanced resistance to Pst in older but not younger leaves. Whereas loss-of-function mutations in Pic12 resulted in enhanced resistance in both older and younger leaves. Overexpression of Pic3 and Pic12 proteins in leaves of Nicotiana benthamiana inhibits resistance to Pst and this effect is dependent on phosphatase activity and an N-terminal palmitoylation motif associated with localization to the cell periphery. Pic3 but not Pic12 has a slight negative effect on flagellin-associated reactive oxygen species generation, although their involvement in the response to Pst appears independent of flagellin. RNA-sequencing analysis of Rio Grande (RG)-PtoR wild-type plants and two independent RG-pic3 mutants revealed that the enhanced disease resistance in RG-pic3 older leaves is associated with increased transcript abundance of multiple defense related genes. RG-pic3/RG-pic12 double mutant plants exhibit stronger disease resistance than RG-pic3 or RG-pic12 single mutants. Together, our results reveal that Pic3 and Pic12 negatively regulate tomato immunity in an additive manner through flagellin-independent pathways.

show abstract

Section: Resultsmentioning

confidence: 99%

Related PP2C phosphatases Pic3 and Pic12 negatively regulate immunity in tomato toPseudomonas syringae

Xia,

Zhang,

Smith

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…Because depletion of suppressed hydrolases is less likely to cause disease phenotypes, we tested whether increased expression could overcome the suppression and uncover the roles of these hydrolases in immunity. We therefore took advantage of the recently developed ‘agromonas’ assay (Buscaill et al ., 2021 ), which is based on infections of agroinfiltrated tissues with Pto DC3000( ΔhQ ). We cloned and transiently expressed the five selected hydrolases through agroinfiltration and infected the agroinfiltrated leaves 2 d later with Pto DC3000( ΔhQ ).…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, we found that transient overexpression of Nb PR3 results in increased immunity to Pto DC3000( ΔhQ ), confirming that supressed hydrolases may act in immunity. Likewise, we found that transient overexpression of BGAL1 increases bacterial resistance (Buscaill et al ., 2021 ). However, not all suppressed hydrolases reduced bacterial growth upon overexpression in our agromonas assay.…”

Section: Discussionmentioning

confidence: 99%

Activity‐based proteomics uncovers suppressed hydrolases and a neo‐functionalised antibacterial enzyme at the plant–pathogen interface

et al. 2023

Self Cite

View full text Add to dashboard Cite

Summary The extracellular space of plant tissues contains hundreds of hydrolases that might harm colonising microbes. Successful pathogens may suppress these hydrolases to enable disease. Here, we report the dynamics of extracellular hydrolases in Nicotiana benthamiana upon infection with Pseudomonas syringae. Using activity‐based proteomics with a cocktail of biotinylated probes, we simultaneously monitored 171 active hydrolases, including 109 serine hydrolases (SHs), 49 glycosidases (GHs) and 13 cysteine proteases (CPs). The activity of 82 of these hydrolases (mostly SHs) increases during infection, while the activity of 60 hydrolases (mostly GHs and CPs) is suppressed during infection. Active β‐galactosidase‐1 (BGAL1) is amongst the suppressed hydrolases, consistent with production of the BGAL1 inhibitor by P. syringae. One of the other suppressed hydrolases, the pathogenesis‐related NbPR3, decreases bacterial growth when transiently overexpressed. This is dependent on its active site, revealing a role for NbPR3 activity in antibacterial immunity. Despite being annotated as a chitinase, NbPR3 does not possess chitinase activity and contains an E112Q active site substitution that is essential for antibacterial activity and is present only in Nicotiana species. This study introduces a powerful approach to reveal novel components of extracellular immunity, exemplified by the discovery of the suppression of neo‐functionalised Nicotiana‐specific antibacterial NbPR3.

show abstract

“…At low bacterial concentrations (OD 600 =0.00001 to 0.0001 for Pto DC3000Q - and OD 600 =0.01 to 0.04 for Agrobacterium), Agrobacterium and Pto DC3000Q - did not interfere with each other, facilitating bacterial pathogen growth assays. Buscaill et al (2021) reported a similar disease assay based on sequential infection of Pto DC3000Q - following Agrobacterium-mediated transient expression in N. benthamiana (Buscaill et al, 2021).…”

Section: Resultsmentioning

confidence: 99%

A conserved glutamate residue in RPM1-interacting protein4 is ADP-ribosylated by Pseudomonas effector AvrRpm2 to activate RPM1-mediated response

Yoon

Middleditch

Rikkerink

2021

Preprint

View full text Add to dashboard Cite

Gram-negative bacterial plant pathogens directly inject effectors into their hosts to hijack and manipulate metabolism, eluding the frontier surveillance at the cell surface. The effector AvrRpm1Pma from Pseudomonas syringae pv. maculicola functions as an ADP-ribosyl transferase, modifying RPM1-interacting protein4 (RIN4), leading to the activation of Arabidopsis resistance protein RPM1. We identified the ADP-ribosyl transferase activity of another bacterial effector AvrRpm2Psa from Pseudomonas syringae pv. actinidiae via infection using a Pseudomonas syringae pv. tomato strain following Agrobacterium-mediated transient expression of RIN4 in N. benthamiana. We conducted mutational analysis in combination with mass spectrometry to genetically locate the modified residue. We show that a conserved glutamate residue (E156) of AtRIN4 is the target site for AvrRpm2Psa by demonstrating the modified AtRIN4 with E156A substitution is no longer ADP-ribosylated. Accordingly, naturally occurring soybean and snap bean RIN4 homologs with no glutamate at the positions corresponding to the E156 of AtRIN4 are not ADP-ribosylated by AvrRpm2Psa. In contrast with another effector AvrB, modifications of potential phosphorylation sites including T166 in AtRIN4 affected neither ADP-ribosylation nor RPM1 activation by AvrRpm2Psa. This study suggests that separate biochemical reactions by different pathogen effectors may trigger the activation of the same resistance protein through distinct modifications of RIN4.

show abstract

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

Abstract: Significance Statement This technical advance introduces a robust and reliable disease assay to monitor bacterial growth of Pseudomonas syringae in agroinfiltrated tissues using an antibiotics cocktail to select against Agrobacterium tumefaciens.

Cited by 21 publications

References 59 publications

Related PP2C phosphatases Pic3 and Pic12 negatively regulate immunity in tomato toPseudomonas syringae

Related PP2C phosphatases Pic3 and Pic12 negatively regulate immunity in tomato toPseudomonas syringae

Activity‐based proteomics uncovers suppressed hydrolases and a neo‐functionalised antibacterial enzyme at the plant–pathogen interface

A conserved glutamate residue in RPM1-interacting protein4 is ADP-ribosylated by Pseudomonas effector AvrRpm2 to activate RPM1-mediated response

Contact Info

Product

Resources

About