University of Stavanger

Peroxisome Targeting of Alternatively Spliced Pathogen Defence Proteins

Center for Organelle Research (CORE), University of Stavanger, Norway

Marit Larsen, Amr Kataya, Chimuka Mwaanga, Kirsti Sørhagen and Sigrun Reumann

ABSTRACT Photorespiration represents one of the major highways of primary plant metabolism and is the most prominent example of metabolic cell organelle integration, since the pathway requires the concerted action of plastidial, peroxisomal, mitochondrial and cytosolic enzymes and organellar transport proteins. Recycling of 2-phosphoglycolate by the photorespiratory C2 cycle is concomitant with stoichiometric production rates of H2O2 in peroxisomes. Apart from its significance for agricultural productivity, a secondary function of photorespiration in pathogen defence has emerged only recently (for review see Sørhagen et al., 2013, Plant Biology 15:723-736). Peroxisomes are highly dynamic ubiquitous eukaryotic organelles involved in numerous processes such as primary and secondary metabolism, development and responses to abiotic and biotic stresses. We screened the Arabidopsis genome for newly predicted proteins targeted to peroxisomes using machine learning methods (Lingner et al., 2011) with focus on homologues of known pathogen defence proteins. Several defence proteins were found to possess yet unknown peroxisome targeting signals (PTS), either as primary subcellular targeting signals or as secondary signals directing alternatively spliced protein variants to peroxisomes under specific (yet unknown) conditions. In the present study we validated peroxisome targeting of two defence proteins by in vivo subcellular targeting analysis. Nudix hydrolases (NUDT) hydrolyse a broad range of different nucleoside diphosphates (linked to some moiety X). NUDT homologs such as NUDT7 play major roles in pathogen defence. The full-length alternative splice variant 2 of nudix hydrolase homologue 15 carrying the predicted PTS1 PKM> (NUDT15.2) was indeed targeted to peroxisomes in transformed onion epidermal cells. Likewise, the protein’s C-terminal decapeptide was sufficient to direct the reporter protein to peroxisomes. By contrast and also fully consistent with the PTS1 protein predictions, EYFP extended by the C-terminal decapeptide of NUDT15 alternative splice variant 1 terminating with CMP> remained cytosolic. Similar subcellular targeting data were obtained for disease resistance protein DRP1, a member of the CC-NBS-LRR class family. Its splice variant 2 was validated to possess a functional PTS1 domain terminating with CRL>, while variant 1 (LMR>) remained cytosolic. Next, we will carry out splice variant specific expression analyses, for instance during Arabidopsis treatment with defence hormones such as SA and JA and during plant infection with virulent and avirulent Pseudomonas strains. Taken together, the analyses further support the idea that the peroxisomal compartment plays multi-faceted roles in pathogen defence beyond metabolism of reactive oxygen species.

Background

References

Acknowledgment

Lingner T, Kataya AR, Antonicelli GE, Benichou A, Nilssen K, Chen XY, Siemsen T, Morgenstern B, Meinicke P, and Reumann S (2011) Identification of novel plant peroxisomal targeting signals by a combination of machine learning methods and in vivo subcellular targeting analyses. Plant Cell 23:1556-1572.

Sørhagen K, Laxa M, Peterhänsel C, Reumann S (2013) The emerging role of photorespiration and non-photorespiratory peroxisomal metabolism in pathogen defence. Plant Biol. 15:723-736.

Chaouch S., Queval G., Vanderauwera S., Mhamdi A., Vandorpe M., Langlois-Meurinne M., Van Breusegem F., Saindrenan P., Noctor G. (2010) Peroxisomal hydrogen peroxide is coupled to biotic defense responses by ISOCHORISMATE SYNTHASE1 in a daylength related manner. Plant Physiol., 153, 1692-1705.

Several pathogen defence proteins have been recently identified in Arabidopsis thaliana that are predicted to be directed to peroxisomes. Peroxisomes are highly dynamic, and are involved in numerous processes in the cell. Plant pathogens use diverse life strategies, and reactive oxygen species (ROS) are major players in the pathogen defence in plants, acting in second message signalling as highly oxidative agents. Peroxisomal proteins have been reported being involved in pathogen defence, where findings have revealed a link between immune responses and genetically defines peroxisomal components (for review see Sørhagen et al., 2013).

CPK1 PEN2

Photorespiration H2O2

Indole glucosinolates

Ca2+

Host defense and/or effector

proteins ?

Redox pertubation

JA JA biosynthesis

O2-, NO (?) O2

- and NO production

Polyamine metabolism

ICS1-dep-. SA synthesis

Cell death, camalexin synthesis,

resistance

LEAF PEROXISOME

SA signaling

Importomer

AGI code Acronym N-term. target. signal

C-term. tripep.

Pred.

AT1G58807.1 DRP1.1 n.p. LMR AT1G58807.2 DRP1.2 n.p. CRL P AT1G59124.1 DRP1.3 n.p. CRL P AT1G28960.1 AtNUDT15.1 mTP (2) CMP AT1G28960.2 AtNUDT15.2 mTP (2) PKM P AT1G28960.3 AtNUDT15.3 mTP (2) CMP AT1G28960.4 AtNUDT15.4 mTP (2) PKM P AT1G28960.5 AtNUDT15.5 mTP (2) CMP

Table I: Multiple models of Arabidopsis genes predicted to express both peroxisomal (PTS1 proteins) and non-peroxisomal variants. N-terminal targeting was predicted by TargetP with high (score=1, given in parenthesis) or low (score=5) confidence. n.p.: not predicted; cTP/mTP: chloroplast/mitochondrial targeting peptide; P: peroxisomal (taken from Sørhagen et al., 2013)

EYFP-DRP1.2(full-length, CRL>) CRL> EYFP DRP1.2

LMR> EYFP Ct 7aa (DRP1.1) EYFP-Ct7aa(DRP1.1, LMR>)

EYFP-Ct7aa(DRP1.2, CRL>) CRL> EYFP Ct 7aa (DRP1.2)

EYFP-NUDT15.2(full-length, PKM>) PKM> EYFP NUDT15.2

CMP> EYFP Ct 7aa (NUDT15.1) EYFP-Ct7aa(NUDT15.1, CMP>):

C. EYFP D1. EYFP

PKM> EYFP Ct 7aa (NUDT15.2) EYFP-Ct7aa(NUDT15.2, PKM>):

Figure 2: In vivo subcellular targeting analysis of the Arabidopsis NUDT15. Onion epidermal cells were transformed biolistically with EYFP-NUDT15 fusion construct. Subcellular targeting was analyzed by fluorescence microscopy after about18 h expression at room temperature only. EYFP-NUDT15 (E1) targeted punctuate subcellular structures (green dots) that were confirmed to be peroxisomes (yellow dots) by using a peroxisomal marker, DsRed-SKL (E2) in merge (E3).

Alternative splice variants evolving PTS1

DRP1 The biggest class of resistance (R) proteins are the nucleotide-binding site leucine-rich repeat (NB-LRR) proteins which are subdivided into two groups based on their N-terminal domain; either the Toll-interleukin-1 receptor (TIR) or coiled-coil (CC) domain. R proteins are important for recognizing bacterial effectors which seek to suppress the plant immune response, initiating plant effector triggered immunity. One R protein belonging to the class of CC-NB-LRRs (At1g58807.2) was identified as having a strongly predicted PTS1 and was named disease resistance protein 1 (DRP1, Lingner et al., 2011). The C-terminal 10 amino acid-peptide of DRP1.1 localized to the cytosol as predicted.

NUDT15

Functional model

The Nudix (NUDX/NUDT) gene family can be found in organisms ranging from bacteria to mammals, and consists of 27 members in Arabidopsis. NUDT15.1 and the splice variant NUDT15.2, both possess an N-terminal mitochondrial targeting signal and have been confirmed mitochondrial, but NUDT15.2 also possesses a potential PTS1 which can direct it to peroxisomes when the mitochondrial signal is blocked by an N-terminal GFP fusion protein. When expressing just the Ct10aa we see cytosolic localization of NUDT15.1 and strong peroxisomal localization (within 24 hours) of NUDT15.2 due to the PTS1 of this splice variant. The protein has in vitro CoA pyrophosphohyrolase activity and may serve a similar function as mammalian NUDT7.

DRP1.1-LMR>

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A. EYFP B1. EYFP B2. DsRed B3. Merge

D2. DsRed D3. Merge

A. EYFP B1. EYFP B2. DsRed B3. Merge

D2. DsRED D1. EYFP D3. Merge C. EYFP

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F1. EYFP EF. Merge E. EYFP

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E. EYFP F2. DsRED F1. EYFP F3. Merge

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F2. DsRED The research has been supported by funding from the Norwegian Research Council and the University of Stavanger.

PTS1/2 PTS1/2

Figure 3: Reported and predicted functions of plant peroxisomes in pathogen defense. Several peroxisome functions have reported links to pathogen defense. The function of peroxisomal H2O2 is presented according to Chaouch et al. (2010). JA, jasmonic acid; NO, nitric oxide; O2-, superoxide anion.

No PTS1 in NUDT15.1

Peroxisome targeting of NUDT15.2

PTS1 in NUDT15.2

No PTS1 in DRP1.1

(weak) peroxisome targeting of DRP1.2

PTS1 DRP1.2

AGI code Acronym Protein coding gene models

AT1G58807.1 DRP1.1

AT1G58807.2 DRP1.2

AT1G59124.1 DRP1.3

AT1G28960.1 AtNUDT15.1

AT1G28960.2 AtNUDT15.2

AT1G28960.3 AtNUDT15.3

AT1G28960.4 AtNUDT15.4

AT1G28960.5 AtNUDT15.5

Figure 1: In vivo subcellular targeting analysis of the Arabidopsis DRP1. Onion epidermal cells were transformed biolistically with EYFP-DRP1.1 or C-terminal domain constructs of DRP1.1 and DRP1.2. Subcellular targeting was analyzed by fluorescence microscopy. EYFP-NUDT15 (E1) targeted punctuate subcellular structures (green dots) that were confirmed to be peroxisomes (yellow dots) by using a peroxisomal marker, DsRed-SKL (E2) in merge (E3).

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