Tomato Prf requires NLR helpers NRC2 and NRC3 to confer resistance against the bacterial speck pathogen Pseudomonas syringae pv. tomato

Bacterial speck, caused by the pathogen Pseudomonas syringae pv. tomato, is one of the most common diseases in tomato production. Together with Pto kinase, the NLR (nucleotide-binding domain leucine-rich repeat containing) protein Prf confers resistance against the bacterial speck pathogen by recognizing AvrPto and AvrPtoB, two Type III effector proteins secreted by P. syringae pv. tomato. This Prf/Pto pathway is part of a complex NLR network in solanaceous plants that mediates resistance to diverse pathogens through the helper NLR proteins NRCs (NLR required for cell death). We previously showed that, in Nicotiana benthamiana, the hypersensitive cell death elicited by expression of AvrPto and Pto, which activate immunity through the endogenous Prf ortholog NbPrf, requires functionally redundant NRC2 and NRC3. However, whether tomato (Solanum lycopersicum) Prf (SlPrf) confers resistance to the bacterial speck pathogen through NRC2 and NRC3 has not been determined. In this study, we show that SlPrf requires NRC2 and NRC3 to trigger hypersensitive cell death and disease resistance in both N. benthamiana and tomato. We found that the hypersensitive cell death induced by AvrPtoB/Pto/SlPrf in N. benthamiana is compromised when NRC2 and NRC3 are silenced, indicating that SlPrf is an NRC2/3-dependent NLR. We validated this finding by showing that silencing NRC2 and NRC3 in the bacterial speck resistant tomato ‘Rio Grande 76R’ compromised Prf-mediated resistance. These results indicate that the NRC network extends beyond N. benthamiana to solanaceous crops.

Regulation of Tomato Prf by Pto-like Protein Kinases

Tomato Prf encodes a nucleotide-binding domain shared by Apaf-1, certain R proteins, and CED-4 fused to C-terminal leucine-rich repeats (NBARC-LRR) protein that is required for bacterial immunity to Pseudomonas syringae and sensitivity to the organophosphate fenthion. The signaling pathways involve two highly related protein kinases. Pto kinase mediates direct recognition of the bacterial effector proteins AvrPto or AvrPtoB. Fen kinase is required for fenthion sensitivity and recognition of bacterial effectors related to AvrPtoB. The role of Pto and its association with Prf has been characterized but Fen is poorly described. We show that, similar to Pto, Fen requires N-myristoylation and kinase activity for signaling and interacts with the N-terminal domain of Prf. Thus, the mechanisms of activation of Prf by the respective protein kinases are similar. Prf–Fen interaction is underlined by coregulatory mechanisms in which Prf negatively regulates Fen, most likely by controlling kinase activity. We further characterized negative regulation of Prf by Pto, and show that regulation is mediated by the previously described negative regulatory patch. Remarkably, the effectors released negative regulation of Prf in a manner dependent on Pto kinase activity. The data suggest a model in which Prf associates generally with Pto-like kinases in tightly regulated complexes, which are activated by effector-mediated disruption of negative regulation. Release of negative regulation may be a general feature of activation of NBARC-LRR proteins by cognate effectors.

Diverse AvrPtoB Homologs from Several Pseudomonas syringae Pathovars Elicit Pto-Dependent Resistance and Have Similar Virulence Activities

ABSTRACT AvrPtoB is a type III effector protein from Pseudomonas syringae pv. tomato that physically interacts with the tomato Pto kinase and, depending on the host genotype, either elicits or suppresses programmed cell death associated with plant immunity. We reported previously that avrPtoB-related sequences are present in diverse gram-negative phytopathogenic bacteria. Here we describe characterization of avrPtoB homologs from P. syringae pv. tomato T1, PT23, and JL1065, P. syringae pv. syringae B728a, and P. syringae pv. maculicola ES4326. The avrPtoB homolog from P. syringae pv. maculicola, hopPmaL, was identified previously. The four new genes identified in this study are designated avrPtoBT1 , avrPtoBPT23 , avrPtoBJL1065 , and avrPtoBB728a . The AvrPtoB homologs exhibit 52 to 66% amino acid identity with AvrPtoB. Transcripts of each of the avrPtoB homologs were detected in the Pseudomonas strains from which they were isolated. Proteins encoded by the homologs were detected in all strains except P. syringae pv. tomato T1, suggesting that T1 suppresses accumulation of AvrPtoBT1. All of the homologs interacted with the Pto kinase in a yeast two-hybrid system and elicited a Pto-dependent defense response when they were delivered into leaf cells by DC3000ΔavrPtoΔavrPtoB, a P. syringae pv. tomato strain with a deletion of both avrPto and avrPtoB. Like AvrPtoB, all of the homologs enhanced the ability of DC3000ΔavrPtoΔavrPtoB to form lesions on leaves of two susceptible tomato lines. With the exception of HopPmaL which lacks the C-terminal domain, all AvrPtoB homologs suppressed programmed cell death elicited by the AvrPto-Pto interaction in an Agrobacterium-mediated transient assay. Thus, despite their divergent sequences, AvrPtoB homologs from diverse P. syringae pathovars have conserved avirulence and virulence activities similar to AvrPtoB activity.