Helper NLRs Nrc2 and Nrc3 act codependently with Prf/Pto and activate MAPK signaling to induce immunity in tomato

Plant intracellular immune receptors, primarily nucleotide-binding, leucine-rich repeat proteins (NLRs), detect pathogen effector proteins and activate NLR-triggered immunity (NTI). Recently, 'sensor' NLRs have been reported to function with 'helper' NLRs to activate immunity. We investigated the role of two helper NLRs, Nrc2 and Nrc3, on immunity in tomato to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) mediated by the sensor NLR Prf and the Pto kinase. An nrc2/nrc3 mutant no longer activated Prf/Pto-mediated NTI to Pst containing the effectors AvrPto and AvrPtoB. An nrc3 mutant showed intermediate susceptibility between wild-type plants and a Prf mutant, while an nrc2 mutant developed only mild disease. These observations indicate that Nrc2 and Nrc3 act additively in Prf-/Pto-mediated immunity. We examined at what point Nrc2 and Nrc3 act in the Prf/Pto-mediated immune response. In the nrc2/3 mutant, programmed cell death (PCD) normally induced by constitutively active variants of AvrPtoB, Pto, or Prf was abolished, but that induced by M3Kα or Mkk2 was not. PCD induced by a constitutively active Nrc3 was also abolished in a Nicotiana benthamiana line with reduced expression of Prf. MAPK activation triggered by expression of AvrPto in the wild-type tomato plants was completely abolished in the nrc2/3 mutant. These results indicate that Nrc2 and Nrc3 act with Prf/Pto and upstream of MAPK signaling. Nrc2 and Nrc3 were not required for PCD triggered by Ptr1, another sensor NLR-mediating Pst resistance, although these helper NLRs do appear to be involved in resistance to certain Pst race 1 strains.