Plant Physiology Preview. Published on June 8, 2011, as DOI:10.1104/pp.111.177501 Running Title: PPR protein PGN in biotic and abiotic stress responses Corresponding author: Tesfaye Mengiste Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 Telephone: 765-494-0599; e-mail: [email protected] Research category: Plants interacting with other organisms 1 Downloaded from on April 4, 2019 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. Copyright 2011 by the American Society of Plant Biologists The Arabidopsis mitochondrial localized pentatricopeptide repeat protein PGN functions in defense against necrotrophic fungi and abiotic stress tolerance 1 Kristin Laluk, Synan AbuQamar2 and Tesfaye Mengiste* Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 Running Title: PPR protein PGN is required for biotic and abiotic stress responses 2 Downloaded from on April 4, 2019 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. 1. This work was supported by the National Science Foundation (grant no. IOB–0749865 to T. M.) 2. Present address: United Emirate University, Faculty of Science, Department of Biology P.O. Box 17551, Al-Ain, United Emirate University. * Corresponding author; e-mail [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org): Tesfaye Mengiste, [email protected]. 3 Downloaded from on April 4, 2019 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. Abstract Pentatricopeptide repeat proteins (PPRPs) are encoded by a large gene family in Arabidopsis and their functions largely unknown. The few studied PPRPs are implicated in different developmental processes through their function in RNA metabolism and post- transcriptional regulation in plant organelles. Here we studied the functions of Arabidopsis PENTATRICOPEPTIDE REPEAT PROTEIN (PPR) FOR GERMINATION ON NaCl (PGN) in plant defense and abiotic stress responses. Inactivation of PGN results in susceptibility to necrotrophic fungal pathogens as well as hypersensitivity to abscisic acid (ABA), glucose, and salinity. Interestingly, ectopic expression of PGN results in the same phenotypes as the pgn null allele indicating a tight regulation of the PGN transcript is required for normal function. Loss of PGN function dramatically enhanced reactive oxygen species (ROS) accumulation in seedlings in response to salt stress. Inhibition of ABA synthesis and signaling partially alleviates the glucose sensitivity of pgn suggesting the mutant accumulates high endogenous ABA. In accordance, induction of NCED3, encoding the rate limiting enzyme in stress-induced ABA biosynthesis, is significantly higher in pgn and the mutant has higher basal ABA levels which may underlie its phenotypes. The pgn mutant has altered expression of other ABA-related genes as well as mitochondrial-associated transcripts, most notably elevated levels of ABI4 and AOX1a known for their roles in retrograde signaling induced by changes in or inhibition of mitochondrial function. These data, coupled with its mitochondrial localization, suggest PGN functions in regulation of ROS homeostasis in mitochondria during abiotic and biotic stress responses, likely through involvement in retrograde signaling. 4 Downloaded from on April 4, 2019 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. Introduction Plants display diverse survival mechanisms against microbial infection and other environmental stresses. While specialized host responses do occur, many components of the molecular events underlying plant responses to abiotic and biotic stresses are common. Passive defenses such as the cuticle aid in drought tolerance and protection from UV damage while also acting as a deterrent of herbivory and barrier against pathogen infection (Reina-Pinto and Yephremov, 2009). Similarly, the cellular and biochemical processes associated with active responses to different abiotic and biotic stimuli also share functional overlaps (Fujita et al., 2006). These induced responses are largely mediated by plant hormones and their interactions which range from simple synergism or antagonism to intricate networks of cross-regulation (Grant and Jones, 2009). Responses to pathogen infection are modulated by salicylate (SA), jasmonate (JA), and ethylene (ET) with a growing role for abscisic acid (ABA), auxin and gibberellins. ABA, a major regulator of environmental stress responses, is generally regarded as a negative regulator of plant defense, with exogenous application or increased endogenous levels typically correlating with plant susceptibility to pathogens (Mauch-Mani and Mauch, 2005; Fujita et al., 2006). However, there are instances of ABA positively contributing to disease resistance through modulation of callose deposition, stomatal closure, defense gene expression, and accumulation of reactive oxygen species (ROS) (Mauch-Mani and Mauch, 2005). Overall, plant responses to different stresses share significant overlap and points of convergence defined by regulatory factors that integrate signaling from various pathways (Fujita et al., 2006; Robert-Seilaniantz et al., 2010). Among these, the Arabidopsis R2R3MYB transcription factor BOS1 is a mediator of abiotic and biotic stress responses, its loss of function resulting in susceptibility to necrotrophic infection as well as hypersensitivity to salt, osmotic, and oxidative stress (Mengiste et al., 2003). Similarly, over-expression of the ATAF1 transcription factor results in susceptibility to Botrytis cinerea and Blumeria graminis f. sp. hordei as well as decreased tolerance to ABA, salt and oxidative stress (Jensen et al., 2007; Wu et al., 2009). PHYTOCHROME AND FLOWERING TIME1 (PFT1) regulates plant resistance to Alternaria brassicicola, B. cinerea, and Fusarium oxysporum through its function in the biosynthesis of anthocyanin, a flavonoid linked to numerous abiotic and biotic stress responses (Kidd et al., 2009). PFT1 encodes a subunit of the evolutionarily conserved Mediator complex which was recently shown to promote transcription of microRNA (miRNA) genes by recruiting 5 Downloaded from on April 4, 2019 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. RNA polymerase II to their promoters (Kim et al., 2011). miRNAs and small interfering RNAs (siRNAs) have emerged as important regulators of plant defense and stress tolerance known to affect gene expression, ROS accumulation, and plant cell death (Borsani et al., 2005; Katiyar- Agarwal et al., 2007; Sunkar et al., 2007; Xie and Qi, 2008). Natural cis-antisense siRNAs (nat- siRNAs) have been associated with Arabidopsis salt tolerance as well as resistance to pathogens (Borsani et al., 2005; Katiyar-Agarwal et al., 2007; Xie and Qi, 2008). Nat-siRNAATGB2 contributes to RPS2-mediated resistance to Pseudomonas syringae by repressing PENTATRICOPEPTIDE REPEAT (PPR) PROTEIN-LIKE (PPRL) gene expression (Katiyar- Agarwal et al., 2007). Members of the eukaryotic pentatricopeptide repeat (PPR) protein (PPRP) family contain tandem arrays of a degenerate 35 amino acid repeat and function in RNA or DNA modification through sequence-specific binding (Saha et al., 2007). As such, PPRPs have been associated with all stages of RNA processing, maturation, and translation (Saha et al., 2007; Schmitz- Linneweber and Small, 2008). PPRPs are classified into subgroups based on C-terminus domains as well as the nature and order of their repeats (Small and Peeters, 2000; Lurin et al., 2004). Three conserved motifs, E-, E+, and DYW, in the C-terminus define four subclasses of the PPRP family. These domains always require the one prior to be present in the protein, therefore the subclasses consist of PPRPs with no C-terminal motifs, E-, E+ (preceded by E-), and DYW (preceded by E+ and E-) (Lurin et al., 2004). The repeat motifs are defined as P, L, or S based on size and variability: P-type is the characteristic repeat defining the protein family, L-type is a long variant of the P repeat, and S-type is a short variant (Lurin et al., 2004). The Arabidopsis genome contains more than 450 PPRPs, yet surprisingly few have been studied and their functions remain largely unknown (Schmitz-Linneweber and Small, 2008). The few studied PPRPs play diverse and crucial roles in plant growth and development including embryogenesis, circadian rhythm, chloroplast development, and retrograde nuclear signaling (Lurin et al., 2004; Oguchi et al., 2004; Tzafrir et al., 2004; Cushing et al., 2005; Ding et al., 2006; Koussevitzky et al., 2007; Chi et al., 2008). GUN1, a DNA-binding chloroplast PPRP, is involved in retrograde signaling, regulation of ABI4 expression and photo-oxidative stress responses (Zhang et al., 2006; Koussevitzky et al., 2007). ABI4 functions in light and sugar-induced stress responses as well as callose-mediated defense, ROS-signaling, and resistance to fungal infection (Ton et al., 2009). LOVASTATIN INSENSITIVE1 (LOI1) is a PPRP that regulates biosynthesis of 6 Downloaded from on April 4, 2019 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. isoprenoids, metabolites known to affect defense gene expression in response to wounding and pathogen infection (Kishimoto et al., 2005; Kobayashi et al., 2007). The loi1 mutant has decreased sensitivity to two inhibitors of isoprenoid synthesis, the fungal phytotoxin lovastatin and the herbicide clomazone, as evidenced by higher sterol and chlorophyll accumulation compared to that of treated wild-type plants (Kobayashi et al., 2007). PPR40 is a mitochondrial PPRP involved in oxidative respiration that also contributes to abiotic stress tolerance in Arabidopsis (Zsigmond et al., 2008). The ppr40 mutant exhibits enhanced sensitivity to ABA and salinity that correlates with increased ROS accumulation and altered stress-responsive gene expression. Thus far, of the 450 predicted PPRPs, only GUN1, LOI1, PPRL, and PPR40 have been associated with Arabidopsis defense and/or stress tolerance (Katiyar-Agarwal et al., 2006; Kobayashi et al., 2007; Koussevitzky et al., 2007; Zsigmond et al., 2008). However, the functional link of many PPRPs in chloroplast and mitochondrial development and/or regulation suggests they may play a role in managing perturbations in cellular redox elicited by different types of stress (Lurin et al., 2004; Andres et al., 2007; Saha et al., 2007). Here we describe the function of the Arabidopsis PENTATRICOPEPTIDE REPEAT PROTEIN (PPR) FOR GERMINATION ON NaCl (PGN) in plant resistance to necrotrophic fungi and tolerance to abiotic stress. Results Identification of the PGN gene and its role in resistance to necrotrophic pathogens Previously, Arabidopsis BOTRYTIS-INDUCED KINASE1 (BIK1) was found to play a contrasting role in plant defense, positively contributing to resistance against B. cinerea but functioning as a negative regulator of resistance against virulent P. syringae (Veronese et al., 2006). In an effort to further define the role of BIK1 in defense and identify genes involved in resistance, we compared the genome-wide transcript profiles of wild-type and bik1 plants prior to and following B. cinerea inoculation (Dhawan et al., 2009). From this, AT1G56570 (designated PPR HYPERSENSITIVE TO GERMINATION ON NaCl, PGN), encoding a pentatricopeptide repeat (PPR) protein (PPRP), was identified as a potential BIK1 target due to its increased basal expression in the mutant and significant induction in wild-type plants following infection (Fig. 1a). Interestingly, Arabidopsis nahG lines have reduced B. cinerea-induced PGN expression (Fig. 1a). nahG plants are SA-deficient whereas bik1 mutation leads to high basal and induced 7 Downloaded from on April 4, 2019 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. SA-accumulation (Delaney et al., 1994; Veronese et al., 2006) suggesting that PGN expression in response to B. cinerea is at least partially dependent on SA levels. Yet, exogenous application of SA does not affect PGN expression whereas treatment with methyl-jasmonate (MeJA) leads to significant induction and the ET precursor 1-aminocyclopropane-1-carboxylic acid (ACC) causes minor suppression (Fig. 1b). Remarkably, PGN expression significantly correlates with the expression of 13 other PPRP genes as well as several genes associated with RNA-synthesis or processing (Supplemental Fig. 1a) (Obayashi et al., 2009). PGN is also highly expressed in dry and imbibed seed tissue as well as the shoot apex throughout development (Supplemental Fig. 1b). To determine the biological relevance of its B. cinerea-induced gene expression, we characterized plants harboring a null T-DNA insertion allele of the PGN gene (SALK_141937) (Fig. 1c). The pgn mutation resulted in enhanced susceptibility to A. brassicicola as evidenced by increased chlorosis and necrosis at the site of inoculation (Fig. 1d). Compared to wild-type, pgn leaves develop significantly larger disease lesions and support increased fungal proliferation (Fig. 1e). The pgn mutant is also susceptible to B. cinerea. Following spray-inoculation, pgn plants display increased chlorosis and tissue maceration 4 days post-inoculation (dpi) that progresses into abundant leaf decay around 7 dpi (Fig. 1f). Despite relatively slow symptom development, fungal growth in the pgn mutant is higher than wild-type just 24 hours after inoculation based on transcript accumulation of the constitutive B. cinerea ActinA gene (Fig. 1g). Additionally, we assayed pgn plants for altered resistance to the bacterial pathogen P. syringae to further clarify PGN-function in BIK1-regulated defense responses and determine if the altered susceptibility of the mutant to necrotrophic infection is a result of hormone-mediated defense antagonism. Plant immune responses to necrotrophic infection are regulated by JA/ET- mediated signaling events known to antagonize SA-dependent defenses associated with biotrophic resistance (Koornneef and Pieterse, 2008). No difference was observed in bacterial growth between wild-type plants and the pgn mutant inoculated with virulent (DC3000) or avirulent (DC3000AvrRpm1) strains of P. syringae (Supplemental Fig. 2). Interestingly, B. cinerea-induced expression of PR-1 and PDF1.2, considered molecular markers of SA- and JA/ET-dependent defense responses, respectively, are not altered in the pgn mutant (Supplemental Fig. 3a, b). Overall, these results suggest the function of PGN in pathogen resistance is specific to necrotrophs and not associated with SA-, JA-, or ET-mediated defenses. 8 Downloaded from on April 4, 2019 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. PGN encodes a pentatricopeptide repeat protein (PPRP) localized to mitochondria PGN is predicted to encode a PPRP (Fig. 2a) with significant homology to proteins from different plant species and Arabidopsis spanning full-length amino acid sequences (Fig. 2b, Supplemental Fig. 4). The PGN protein shows the characteristics of the E+ PPRP subclass with a 43-S-P-L-S-P-L-S-34-L-S-1-P-L2-S-4-E-E+-19 repeat and motif arrangement (numbers indicate the amount of residues in between motifs, Fig. 2a). The repeats are predicted to form a super- α helix compromised of two anti-parallel -helices able to bind DNA or RNA with substrate- specificity dependent on the residue properties within the groove (Delannoy et al., 2007). In Arabidopsis, there are more than 450 predicted PPRPs, with all described proteins localized to mitochondria, chloroplast, or nucleus (Small and Peeters, 2000; Lurin et al., 2004; Schmitz- Linneweber and Small, 2008). Of the E+ PPRP subfamily, more than 40% are predicted to localize to mitochondria (Lurin et al., 2004). Prediction programs indicate PGN has a cleavable targeting sequence (MSITKLARSNAFKPIPNFVRSSLRN) and a 96% likelihood of subcellular localization to mitochondria (Carlos and Vincens, 1996). To experimentally determine the subcellular localization of PGN, 35S:PGN-GFP was transiently expressed in Nicotiana benthamiana leaves in conjunction with different cellular markers. PGN-GFP co-localized with a mitochondrial marker (mCHERRY) consistent with the in silico predictions (Fig. 2c). Loss or gain of PGN function causes hypersensitivity to ABA, NaCl and glucose To further study the function of PGN, over-expression (35S:PGN;pgn) and complementation lines (PGNpr:PGN;pgn) were generated (Fig. 3a). The different PGN genotypes were assayed for altered responses to hormones and abiotic stress agents in an effort to clarify the mechanism of PGN function in Arabidopsis defense. The pgn mutant displays germination hypersensitivity to media supplemented with ABA and increased glucose (Fig. 3b- e). Over-expression of PGN also results in hypersensitivity to ABA and glucose comparable to that of the mutant (Fig. 3b-e). Similarly, the pgn mutant and over-expression lines have increased sensitivity to salt, displaying increased salt-induced necrosis and chlorosis as well as reduced germination and root growth compared to wild-type seedlings (Fig. 4). Alternatively, both pgn and 35S:PGN;pgn plants have enhanced growth on MS media lacking glucose relative to wild- type (Fig. 3b). In general, transformation with genomic PGN driven by its native promoter (PGNpr:PGN;pgn) restores mutant sensitivity to wild-type levels (Fig. 3b-e and Fig. 4a-d). 9 Downloaded from on April 4, 2019 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. However, at higher concentrations of ABA, salt, and glucose, PGNpr:PGN;pgn seedlings have significantly reduced leaf emergence, root growth, and smaller cotyledons, respectively, relative to wild-type (Fig. 3c-e and Fig. 4a,c,d). This disparity could be attributed to the slightly higher PGN expression of these lines (Fig. 3a) compared to wild-type and further supports the hypersensitivity exhibited by 35S:PGN;pgn plants. Overall, these data suggest that, in addition to defense against necrotrophic infection, PGN regulates plant responses to abiotic stress and its expression level is an important determinant of function. No altered seedling growth was observed when pre-germinated 5-day seedlings were transferred to media supplemented with glucose, ABA or IAA (Supplemental Fig. 5a-c). Also, germination on media supplemented with ACC, MeJA, SA, GA, IAA, or hydrogen peroxide is not affected by the pgn mutation limiting the role of PGN to a specific subset of stress and hormone responses (Supplemental Fig. 5d). The glucose hypersensitivity of the pgn mutant is partially restored by ABA antagonists Increased concentrations of glucose are known to delay Arabidopsis germination, with ABA levels determining the severity of inhibition (Gazzarrini and McCourt, 2001; Dekkers et al., 2004). Many mutants exhibiting enhanced growth on media containing high exogenous sugars are also ABA-insensitive whereas even minimal ABA increases act additively to sugar- mediated seedling growth inhibition (Laby et al., 2000; Gazzarrini and McCourt, 2001; Gibson et al., 2001). To determine if pgn hypersensitivity to glucose and ABA is a result of increased endogenous ABA, we assayed growth responses to 6% glucose in the presence of two ABA inhibitors: the ET-precursor ACC and norflurazon (NF). ET antagonizes ABA function in germination, likely promoting emergence through suppression of ABA-signaling and synthesis initiated by glucose and ABA (Beaudoin et al., 2000; Ghassemian et al., 2000; Leon and Sheen, 2003; Matilla and Matilla-Vazquez, 2008). NF inhibits ABA-accumulation through disruption of carotenoid biosynthesis upstream of ABA-biosynthesis (Bartels and Watson, 1978; Zeevaart and Creelman, 1988). Addition of ACC or NF partially relieved pgn hypersensitivity to glucose relative to 6% glucose alone (Fig. 5a). Interestingly, whereas ACC restored pgn growth to a degree comparable to respective wild-type controls, the effect of NF resulted in seedlings nearly half the size of corresponding wild-type. These data suggest the increased glucose sensitivity exhibited by the pgn mutant is likely due to high levels of endogenous ABA. Subsequent analysis of total ABA content indicates that pgn seedlings do have significantly higher basal 10 Downloaded from on April 4, 2019 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved.