Nα-acetyltransferase NAA50 mediates plant immunity independent of the Nα-acetyltransferase A complex.

In humans and plants, 40% of the proteome is co-translationally acetylated at the N-terminus by a single Nα-acetyltransferase (Nat) termed NatA. The core NatA complex is comprised of the catalytic subunit Nα- acetyltransferase 10 (NAA10) and the ribosome-anchoring subunit NAA15. The regulatory subunit Huntingtin Yeast Partner K (HYPK) and the acetyltransferase NAA50 join this complex in humans. Even though both are conserved in Arabidopsis (Arabidopsis thaliana), only AtHYPK is known to interact with AtNatA. Here we uncover the AtNAA50 interactome and provide evidence for the association of AtNAA50 with NatA at ribosomes. In agreement with the latter, a split-luciferase approach demonstrated close proximity of AtNAA50 and AtNatA in planta. Despite their interaction, AtNatA/HYPK and AtNAA50 exerted different functions in vivo. Unlike NatA/HYPK, AtNAA50 did not modulate drought-tolerance or promote protein stability. Instead, transcriptome and proteome analyses of a novel AtNAA50-depleted mutant (amiNAA50) implied that AtNAA50 negatively regulates plant immunity. Indeed, amiNAA50 plants exhibited enhanced resistance to oomycetes and bacterial pathogens. In contrast to what was observed in NatA-depleted mutants, this resistance was independent of an accumulation of salicylic acid prior to pathogen exposure. Our study dissects the in vivo function of the NatA interactors HYPK and NAA50 and uncovers NatA-independent roles for NAA50 in plants.

Signal Peptide Peptidase and PI4Kß1/2 play opposite roles in plant ER stress response and immunity.

The Arabidopsis pi4kß1,2 mutant is mutated in the phosphatidylinositol 4-kinase (PI4K) ß1 and PI4Kß2 enzymes which are involved in the biosynthesis of phosphatidylinositol 4-phosphate (PI4P), a minor membrane lipid with important signaling roles. pi4kß1,2 plants display autoimmunity and shorter roots. Though the pi4kß1,2 mutant has been extensively characterized, the source of its autoimmunity remains largely unknown. In this study, through a genetic suppressor screen, we identified multiple partial loss-of-function alleles of signal peptide peptidase (spp) that can suppress all the defects of pi4kß1,2. SPP is an intramembrane cleaving aspartic protease. Interestingly, pi4kß1,2 plants display enhanced ER stress response and mutations in SPP can suppress such phenotype. Furthermore, reduced ER stress responses were observed in the spp single mutants. Overall, our study reveals a previously unknown function of PI4Kß and SPP in ER stress and plant immunity.

Deletions within intronic T-DNA lead to reversion of T-DNA mutant phenotypes.

Agrobacterium-mediated transformation enables random transfer-DNA (T-DNA) insertion into plant genomes. T-DNA insertion into a gene's exons, introns or untranscribed regions close to the start or stop codon can disrupt gene function. Such T-DNA mutants have been useful for reverse genetics analysis, especially in Arabidopsis thaliana. As T-DNAs are inserted into genomic DNA, they are generally believed to be stably inherited. Here, we report a phenomenon of reversion of intronic T-DNA mutant phenotypes. From a suppressor screen using intronic T-DNA pi4kß1,2 double mutant, we recovered intragenic mutants of pi4kß1, which suppressed the autoimmunity of the double mutant. These mutants carried deletions in the intronic T-DNAs, resulting in elevated transcription of normal PI4Kß1. Such reversion of T-DNA insertional mutant phenotype stresses the need for caution when using intronic T-DNA mutants and reiterates the importance of using irreversible null mutant alleles in genetic analyses.

Arabidopsis Tubby domain-containing F-box proteins positively regulate immunity by modulating PI4Kß protein levels.

The Tubby domain, named after the TUBBY protein in mice, binds to phosphatidylinositol 4,5-bisphosphate. Arabidopsis has 11 Tubby domain-containing proteins referred to as Tubby-Like Proteins (TLPs). Of the 11 TLPs, 10 possess the N-terminal F-box domain, which can interact with SKP-like proteins and form SKP1-Cullin-F-box E3 ligase complexes. Although mice TUBBY has been extensively studied, plant TLPs' functions are scarcely detailed. In this study, we show that the Arabidopsis Tubby-like protein 6 (TLP6) and its redundant homologs, TLP1, TLP2, TLP5, and TLP10, positively regulate Arabidopsis immune responses. Furthermore, in an immunoprecipitation mass spectrometry analysis to search for ubiquitination substrates of the TLPs, we identified two redundant phosphoinositide biosynthesis enzymes, phosphatidylinositol 4-kinase ß proteins (PI4Kßs), PI4Kß1 and PI4Kß2, as TLP interactors. Importantly, TLP6 overexpression lines fully phenocopy the phenotypes of the pi4kß1,2 mutant, while TLP6 overexpression also leads to increased PI4Kß2 ubiquitination and reduction in its protein level in a proteasome-dependent manner. Most significantly, TLP6 overexpression does not further enhance the autoimmunity of the pi4kß1,2 double mutant, supporting the hypothesis that TLP6 targets the PI4Kßs for ubiquitination and degradation. Thus, our study reveals a novel mechanism where TLPs promote plant immune responses by modulating the PI4Kßs protein levels.

The proteasome regulator PTRE1 contributes to the turnover of SNC1 immune receptor.

Plants have evolved a sophisticated immune system in order to recognize and respond to microbes in their environments. Nucleotide-binding leucine-rich repeat (NLR) proteins detect the presence of specific effector molecules delivered into host cells by pathogens and activate strong defence responses. However, as excessive accumulation of NLRs can result in inappropriate immune responses, their abundance must be tightly regulated. Targeted degradation of NLRs through the ubiquitin proteasome pathway is an important mechanism to limit NLR accumulation. Mutations that perturb NLR degradation can cause autoimmune phenotypes. In this study, we show that the proteasome regulator PTRE1 also contributes to NLR degradation. ptre1 mutant plants exhibit increased defence marker gene expression and enhanced disease resistance against virulent pathogens. The stability of the NLR, SUPPRESSOR OF npr1-1 CONSTITUTIVE 1 (SNC1) is also increased in the ptre1 mutant. Although the mouse homologue of PTRE1 was reported to interact with a Cell Division Control protein 48 (CDC48) homologue in vitro (Clemen et al., 2015), we only observed interaction between PTRE1 and AtCDC48A in a split luciferase assay, but not in co-immunoprecipitation. In addition, a related Arabidopsis protein PTRE1h shares partial redundancy with PTRE1. Together, PTRE1 acts as a negative regulator of plant immunity partly by facilitating the degradation of immune receptors such as SNC1.

MAP kinase signalling: interplays between plant PAMP- and effector-triggered immunity.

In plants, mitogen-activated protein kinase (MAPK) cascades are involved in regulating many biological processes including immunity. They relay signals from membrane-residing immune receptors to downstream components for defense activation. Arabidopsis MPK3/6 and MPK4 are activated in two parallel MAPK cascades during PAMP-triggered immunity. MPK3/6 have been implicated in the activation of various immune responses and their inactivation leads to compromised defense against pathogens. On the other hand, the MEKK1-MKK1/2-MPK4 cascade plays critical roles in basal resistance. Disruption of this MAPK cascade results in constitutive defense responses mediated by the NB-LRR protein SUMM2. Interestingly, SUMM2 guards the MEKK1-MKK1/2-MPK4 cascade activity indirectly through monitoring the phosphorylation status of CRCK3, which is a substrate of MPK4. From the pathogens' side, a number of effectors are shown to target various components of MAPK cascades in plants. Inactivation of MPK4 by the Pseudomonas effector HopAI1 triggers SUMM2-mediated immunity. Together, these findings suggest intricate interplays between PAMP-triggered immunity and effector-triggered immunity via MAPK signaling.