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In plants and animals, nucleotide-binding and leucine-rich repeat domain containing (NLR) immune receptors are utilized to detect the presence or activities of pathogen-derived molecules. However, the mechanisms by which NLR proteins induce defense responses remain unclear. Here, we report the characterization of one basic Helix-loop-Helix (bHLH) type transcription factor (TF), bHLH84, identified from a reverse genetic screen. It functions as a transcriptional activator that enhances the autoimmunity of NLR mutant snc1 (suppressor of npr1-1, constitutive 1) and confers enhanced immunity in wild-type backgrounds when overexpressed. Simultaneously knocking out three closely related bHLH paralogs attenuates RPS4-mediated immunity and partially suppresses the autoimmune phenotypes of snc1, while overexpression of the other two close paralogs also renders strong autoimmunity, suggesting functional redundancy in the gene family. Intriguingly, the autoimmunity conferred by bHLH84 overexpression can be largely suppressed by the loss-of-function snc1-r1 mutation, suggesting that SNC1 is required for its proper function. In planta co-immunoprecipitation revealed interactions between not only bHLH84 and SNC1, but also bHLH84 and RPS4, indicating that bHLH84 associates with these NLRs. Together with previous finding that SNC1 associates with repressor TPR1 to repress negative regulators, we hypothesize that nuclear NLR proteins may interact with both transcriptional repressors and activators during immune responses, enabling potentially faster and more robust transcriptional reprogramming upon pathogen recognition.

Plant defense responses need to be tightly regulated to prevent auto-immunity, which is detrimental to growth and development. To identify negative regulators of Resistance (R) protein-mediated resistance, we screened for mutants with constitutive defense responses in the npr1-1 background. Map-based cloning revealed that one of the mutant genes encodes a conserved TPR domain-containing protein previously known as SRFR1 (SUPPRESSOR OF rps4-RLD). The constitutive defense responses in the srfr1 mutants in Col-0 background are suppressed by mutations in SNC1, which encodes a TIR-NB-LRR (Toll Interleukin1 Receptor-Nucleotide Binding-Leu-Rich Repeat) R protein. Yeast two-hybrid screens identified SGT1a and SGT1b as interacting proteins of SRFR1. The interactions between SGT1 and SRFR1 were further confirmed by co-immunoprecipitation analysis. In srfr1 mutants, levels of multiple NB-LRR R proteins including SNC1, RPS2 and RPS4 are increased. Increased accumulation of SNC1 is also observed in the sgt1b mutant. Our data suggest that SRFR1 functions together with SGT1 to negatively regulate R protein accumulation, which is required for preventing auto-activation of plant immunity.

Plant Resistance (R) proteins play an integral role in defense against pathogen infection. A unique gain-of-function mutation in the R gene SNC1, snc1, results in constitutive activation of plant immune pathways and enhanced resistance against pathogen infection. We previously found that mutations in MOS4 suppress the autoimmune phenotypes of snc1, and that MOS4 is part of a nuclear complex called the MOS4-Associated Complex (MAC) along with the transcription factor AtCDC5 and the WD-40 protein PRL1. Here we report the immuno-affinity purification of the MAC using HA-tagged MOS4 followed by protein sequence analysis by mass spectrometry. A total of 24 MAC proteins were identified, 19 of which have predicted roles in RNA processing based on their homology to proteins in the Prp19-Complex, an evolutionarily conserved spliceosome-associated complex containing homologs of MOS4, AtCDC5, and PRL1. Among these were two highly similar U-box proteins with homology to the yeast and human E3 ubiquitin ligase Prp19, which we named MAC3A and MAC3B. MAC3B was recently shown to exhibit E3 ligase activity in vitro. Through reverse genetics analysis we show that MAC3A and MAC3B are functionally redundant and are required for basal and R protein-mediated resistance in Arabidopsis. Like mos4-1 and Atcdc5-1, mac3a mac3b suppresses snc1-mediated autoimmunity. MAC3 localizes to the nucleus and interacts with AtCDC5 in planta. Our results suggest that MAC3A and MAC3B are members of the MAC that function redundantly in the regulation of plant innate immunity.

Plant Resistance (R) proteins play an integral role in defense against pathogen infection. A unique gain-of-function mutation in the R gene SNC1, snc1, results in constitutive activation of plant immune pathways and enhanced resistance against pathogen infection. We previously found that mutations in MOS4 suppress the autoimmune phenotypes of snc1, and that MOS4 is part of a nuclear complex called the MOS4-Associated Complex (MAC) along with the transcription factor AtCDC5 and the WD-40 protein PRL1. Here we report the immuno-affinity purification of the MAC using HA-tagged MOS4 followed by protein sequence analysis by mass spectrometry. A total of 24 MAC proteins were identified, 19 of which have predicted roles in RNA processing based on their homology to proteins in the Prp19-Complex, an evolutionarily conserved spliceosome-associated complex containing homologs of MOS4, AtCDC5, and PRL1. Among these were two highly similar U-box proteins with homology to the yeast and human E3 ubiquitin ligase Prp19, which we named MAC3A and MAC3B. MAC3B was recently shown to exhibit E3 ligase activity in vitro. Through reverse genetics analysis we show that MAC3A and MAC3B are functionally redundant and are required for basal and R protein-mediated resistance in Arabidopsis. Like mos4-1 and Atcdc5-1, mac3a mac3b suppresses snc1-mediated autoimmunity. MAC3 localizes to the nucleus and interacts with AtCDC5 in planta. Our results suggest that MAC3A and MAC3B are members of the MAC that function redundantly in the regulation of plant innate immunity. [ABSTRACT FROM AUTHOR]