N-terminal modifications contribute to flowering time and immune response regulations

Abstract

A variety of N-terminal co-translational modifications play crucial roles in many cellular processes across eukaryotic organisms. Recently, N-terminal acetylation has been proposed as a regulatory mechanism for the control of plant immunity. Analysis of an N-terminal acetyltransferase complex A (NatA) mutant, naa15–1, revealed that NatA controls the stability of immune receptor Suppressor of NPR1, Constitutive 1 (SNC1) in an antagonistic fashion with NatB. Here, we further report on an antagonistic regulation of flowering time by NatA and NatB, where naa15–1 plants exhibit late flowering, opposite of the early flowering phenotype previously observed in natB mutants. In addition, we provide evidence for the involvement of another N-terminal modification, N-myristoylation, in controlling pathogen-associated molecular pattern (PAMP) triggered immunity (PTI) through the characterization of N-myristoyltransferase 1 (NMT1) defective mutants, which express a low level of NMT1 protein. The mutant line lacks induced production of reactive oxygen species and MAP kinase phosphorylation in response to treatment with the known immune elicitor flg22. NMT1 deficient plants also exhibit increased susceptibility to Pst hrcC, a non-pathogenic Pseudomonas syringae tomato strain lacking a functional type-III secretion system. The potential for the NatA-NatB antagonistic relationship to exist outside of the regulation of SNC1 as well as the disclosing of NMT1s role in PTI further supports the significant contribution of N-terminal co-translational modifications in the regulation of biological processes in plants, and present interesting areas for further exploration.

Post-translational and co-translational protein modifications play critical roles in determining the levels to which a protein accumulates and often effects protein function. In eukaryotes, critical modifications often occur at the N-termini of newly synthesized polypeptides. This has led to the development of what is termed the universal N-end rule, a system used to estimate the half-life of a protein based on its N-terminal residues. The N-end rule describes the various modifications occurring at the N-termini, which often require N-terminal Met excision (NME) followed by modification of the second residue. One of the most common N-terminal modifications seen in eukaryotes is N-terminal acetylation, which is carried out by a class of 6 N-terminal acetyltransferases (Nats), from NatA to NatF.¹ The role of N-terminal acetylation still remains ambiguous although it has been implicated in a range of functions, including protecting proteins from degradation, targeting proteins to membranes, and marking proteins for degradation.¹

Immunity in plants is mediated by an array of receptors which recognize pathogen associated signals and trigger defense locally, and later throughout the plant, in order to defend against pathogen infections.² Components of these pathways require tight regulation since underactive immune signaling would lead to susceptibility while plants may suffer autoimmunity if defense is overactive. Recently a forward genetic screen searching for mutants effecting immune regulation in Arabidopsis identified naa15–1, which encodes the yeast Naa15 ortholog, the auxiliary subunit of NatA.³ The naa15–1 mutant can significantly enhance the autoimmune phenotypes of snc1 (suppressor of npr1, constitutive 1), which carries a gain-of-function mutation in a TIR-type nucleotide binding leucine-rich repeat (TNL) immune receptor. The enhanced immune phenotype was also apparent in the naa15–1 single mutant, seen via dwarfism, enhanced defense marker Pathogenesis-Related (PR) gene expression, and resistance to the virulent oomycete pathogen Hyaloperonospora arabidopsidis (H.a.) Noco2.³

Through examination of protein levels as well as N-terminal acetylation events of SNC1 in the naa15–1 mutant background, the first methionine (Met) residue of SNC1 was found to be targeted by NatA, which marks the protein for degradation. It was also discovered that SNC1 undergoes alternative translational initiation from the second Met and that this second Met is acetylated by NatB. Interestingly, SNC1 accumulates less in natB mutant plants. Consistently, natB can partially suppress snc1, suggesting that acetylation by NatB acts to stabilize SNC1.³ This antagonistic nature of the regulatory effects of NatA and NatB on SNC1 had never been reported before and presents an interesting mode of complex regulation, which could be used to regulate other proteins exhibiting different N-termini during their life-cycle.

In addition to immune defects, naa15–1 plants also exhibit a distinct late flowering phenotype (Fig. 1), which is opposite to the early flowering phenotype seen in natB mutants.⁴ This inverted flowering time phenotype seen between the natA and natB mutants mirrors what was observed in the regulation of plant immunity and suggests that perhaps N-terminal acetylation regulates flowering time in a similarly antagonistic fashion. Although the late flowering phenotype in naa15–1 can be suppressed by a mutation in the master regulator FLC, FLC is not itself a predicted target for N-terminal acetylation according to an efficient prediction tool (TermiNator3; http://www.isv.cnrs-gif.fr/terminator3).⁵ Thus, it is unlikely that FLC is regulated in a similar fashion as SNC1, unless, like was the case with SNC1, the starting position of FLC is incorrectly predicted. How NatA and NatB regulate flowering time will be an interesting question to pursue further.

Figure 1.

naa15–1 plant flowers late and this late flowering phenotype can be partially suppressed by flc-2. (A) Morphology of 5-week-old soil-grown plants of WT, naa15–1, naa15–1 flc-2, and flc-2. (B) The number of rosette leaves of plants of the indicated genotypes counted when the stem is 3–7 cm long. Bar represent means of 12 replicates ±SD.

Acetylation is likely not the only N-terminal modification that plays a role in the regulation of plant immunity or flowering time. It has been suggested that another crucial N-terminal modification, N-myristoylation (MYR), is important for post-embryonic stages including flowering and innate immunity.^6,7 MYR is a protein modification occurring on N-terminal glycine residues upon removal of the first Met. It is known to be essential for specific protein-protein interactions and more generally for targeting proteins to specific membranes.⁸ Many proteins involved in immunity are predicted MYR targets (http://www.isv.cnrs-gif.fr/recherche/tm/maturation/myristoylome2007am.htm). Reduced accumulation of several myristoylated disease resistance proteins and calcium-dependent protein kinases (CPKs) at the plasma membrane (PM) was previously observed in another partial loss-of-function NMT1 mutant.⁷ However, although many proteins involved in PTI localize to the PM, the exact contribution of MYR to PTI is not established.

Because knocking out N-myristoyltransferase 1 (NMT1) is lethal, in order to test the contribution of NMT1 to PTI, it was necessary to use a knockout mutant complemented with a very low expression NMT1 transgene, this line was named F11.⁶ F11 exhibits a variety of phenotypes including lesioning of the rosette leaves and heightened expression of PR genes, suggesting NMT1 plays a negative role in immune responses.^6,9 Here, a close examination of the PTI phenotypes of F11 plants revealed a positive role of MYR in PTI. Key responses involved in PTI include induced reactive oxygen species (ROS) production and phosphorylation of mitogen activated protein kinases (MAPKs). As shown in Figure 2A, upon treatment with flg22, a known elicitor of PTI, no visible elicitation of ROS was seen in F11 plants. Almost no increase in MAPK phosphorylation was visible either (Fig. 2B). To test whether the observed PTI defects correlate with enhanced susceptibility, F11 plants were challenged with Pseudomonas syringae pv. tomato DC3000 hrcC (Pst hrcC). Pst hrcC lacks a component of the type III secretion system and is thus non-pathogenic, allowing only mutants with significant PTI defects to show enhanced growth. The susceptibility of F11 to Pst hrcC confirms the PTI deficiency observed in both the ROS and MAPK responses and demonstrates that MYR plays a positive role in PTI. This effect could be partly explained by the reduced accumulation of CPKs or BIK1 (botrytis-induced kinase 1) to the PM, as BIK1 is predicted to undergo myristoylation and plays a key role in transducing signals immediately downstream of PAMP receptors.^10,11 Both BIK1 and CPK5 were shown to associate with the PM^12,13 and have been shown to directly activate RBOHD (NADPH/respiratory burst oxidase protein D) for ROS production.^14-16 The lack of ROS production could be due to inefficient PM localization of both BIK1 and CPK5, or the result of an unknown MYR target. N-terminal myristoylation adds a new element to immune regulation with the potential to affect a range of immune associated proteins that are targeted to the PM for defense signaling.

Figure 2.

PTI Defects in NMT1 mutant F11 plants. (A) flg22 induced production of ROS in WT and F11 plants. Leaf slices collected from 4-week-old plants were incubated overnight in H₂O after which they were treated with 50 nM flg22 prior to ROS measurement. ROS was measured using a luminol-based assay with measurements taken at 2 minute intervals. Shown are mean values of 12 replicates with error bars representing SD. The experiment was repeated 3 times with similar results. (B) Western blot showing flg22 induced phosphorylation of MAPKs in WT and F11 plants. Twelve-day-old seedlings were grown on ½ MS plates, treated with 1 µM flg22 and harvested at 0 minutes, 10 minutes and 20 minutes post flg22 treatment. MAPK3, MAPK4 and MAPK6 were detected on a western blot using an anti-ERK1/2 antibody specific to the phosphorylated MAPKs. The experiment was repeated twice with similar results. (C) Pst HrcC infection on WT and F11 plants. Leaves of 4-week-old plants were infiltrated with Pst HrcC bacteria (OD₆₀₀ = 0 .005) and samples were collected at 0 and 3 d post inoculation. Included as a positive control is the known susceptible mutant agb1. Data were analyzed using one-way ANOVA, with letters representing statistically significant differences (p-Value < 0.001). Day 0 values are means of 4 replicates and day 3 values are means of 6 replicates, both with error bars representing SD. This experiment was repeated 3 times with similar results.

The complex nature of the regulation seen in the case of SNC1 presents an interesting example but it is difficult to know if this type of antagonistic regulation is unique or widespread. The identification of a second phenotype, flowering time, which may appear to share this antagonistic regulatory relationship suggests that it may in fact be more widespread than anticipated. In addition the newly characterized role of myristoylation in PTI suggests that a great deal of inquiry is still necessary into all forms of N-terminal modifications and what effect they may have on plant immunity.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.