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

Abstract

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.

PP2C phosphatase Pic14 negatively regulates tomato Pto/Prf-triggered immunity by inhibiting MAPK activation

“…Phenotypic evaluation of plants inoculated with Pst DC3000Δ avrPtoB demonstrated that RG-pic14 mutants are more resistant to bacterial infection in their older but not younger leaves. In a related study, we observed a similar phenotype in plants carrying mutations in two PP2Cs sharing sequence similarity, Pic3 and Pic12 (Xia et al, 2024). In that work, RNA-seq analysis revealed that older leaves of RG-pic3 had significantly more differentially expressed genes related to defense compared to younger leaves.…”

“…In a related study we found that two closely related PP2Cs, Pic3 and Pic12, play a partially redundant role in non-NTI plant immune responses (Xia et al, 2024). Pic14 is also related to another PP2C, Pic6 (Solyc05g052520), with both PP2Cs residing in clade E (Qiu et al, 2022) or clade B (Sobol et al, 2022) depending on the phylogenetic analysis and sharing 55% amino acid sequence identity.…”

“…Through an analysis of transcriptomic data of tomato, we identified a subset of PP2C-encoding genes whose transcript abundance increases during various immune responses (Sobol et al, 2022); we now refer to these as P P2C immunity-associated c andidate (Pic) genes. Previously, we showed that Pic1 suppresses pattern-triggered immunity by dephosphorylating the Pti1b kinase (Giska & Martin, 2019; Schwizer et al, 2017) and that Pic3 and Pic12 negatively regulate tomato defense in flagellin independent manner (Xia et al, 2024). Here, we build on the transcriptomic data analysis by Sobol et al (2022) which revealed that 25 out of the 97 PP2C-encoding genes in tomato show differential expression during NTI.…”