‘Candidatus Liberibacter asiaticus’ peroxiredoxin (LasBCP) suppresses oxylipin-mediated defense signaling in citrus

Journal of Plant Physiology 236 (2019) 61–65

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‘Candidatus Liberibacter asiaticus’ peroxiredoxin (LasBCP) suppresses oxylipin-mediated defense signaling in citrus Mukesh Jain, Alejandra Munoz-Bodnar, Dean W. Gabriel

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Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: Citrus greening Huanglongbing Innate immunity Liberibacter Lipid peroxidation Oxylipins Peroxidase Peroxiredoxin RES ROS

The Lasbcp (CLIBASIA_RS00445) 1-Cys peroxiredoxin gene is conserved among all 13 sequenced strains of Candidatus Liberibacter asiaticus, the causal agent of Huanglongbing or "citrus greening" disease. LasBCP was previously characterized as a secreted peroxiredoxin with substrate specificity for organic peroxides, and as a potential pathogenicity effector. Agrobacterium-mediated transient expression of LasBCP in citrus leaves provided significant protection against peroxidation of free and membrane-bound lipids, thereby preserving the molecular integrity of the chlorophyll apparatus and reducing accumulation of lipid peroxidation products (oxylipins) following exposure to tert-butyl hydroperoxide (tBOOH, an organic peroxide). Oxylipins extracted from GUS-expressing citrus leaves reduced viability of L. crescens, the only Liberibacter species cultured to date. However, similar extracts obtained from LasBCP-expressing leaves were less inhibitory to L. crescens growth and viability in culture. Quantitative RT-PCR analyses showed coordinated transcriptional downregulation of oxylipin biosynthetic (CitFAD, CitLOX, CitAOS and CitAOC), and jasmonic acid (JA) (CitJAR1, CitCOI1 and CitJIN1) and salicylic acid (SA) (CitPAL, CitICS and CitPR1) signaling pathway genes in citrus leaves expressing LasBCP and treated with tBOOH. The negative response regulator of jasmonic acid CitJAZ1 was upregulated in LasBCPexpressing citrus leaves under similar conditions. These data clearly demonstrated a protective role of secreted LasBCP in favor of Las survival and colonization by alleviating ROS-induced lipid peroxidation in citrus host, preventing accumulation of antimicrobial oxylipins, and suppressing both localized and systemic immune responses in planta.

1. Introduction Huanglongbing (HLB) or "citrus greening" is arguably the single most devastating citrus disease worldwide. HLB is associated with three fastidious and uncultured α-proteobacteria; Candidatus Liberibacter asiaticus (Las) in Asia and the Americas, Ca. L. americanus in Brazil, and Ca. L. africanus in Africa. The term ‘Candidatus’ is used to describe putative prokaryotic species that have not been isolated, characterized and stored in a culture collection. Advances in metagenomics techniques have allowed determination of complete genome sequences of all three Ca. Liberibacter species associated with HLB. Las is vectored and transmitted among citrus trees by the Asian citrus psyllid Diaphorina citri. Las colonization in citrus is phloem-limited, significantly compromising photoassimilate partitioning and nutrient transport functions. HLB symptoms include leaf mottling, vein corking, stunted growth, lopsided and discolored fruits, premature fruit drop, progressive decline and death of infected trees. To date, all efforts to obtain sustained growth of Las in axenic cultures have failed. Molecular

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characterization of Las pathogenesis is urgently needed to develop effective HLB management strategies (Clark et al., 2018; Jain et al., 2015, 2018; Li et al., 2017). Following pathogen perception, plants activate specific defense signaling cascades resulting in the activation of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), effector-triggered immunity (ETI), and systemic acquired resistance (SAR) (Boutrot and Zipfel, 2017). Compatible host-pathogen interactions are marked by low-amplitude, nonspecific apoplastic bursts of Reactive Oxygen Species (ROS; primarily H2O2) that are mediated through the activity of plant Respiratory Burst Oxidase Homologs (RBOHs) (Kadota et al., 2015). A second high-amplitude oxidative burst occurs during incompatible host-pathogen interactions, resulting in effective disease resistance characterized by a hypersensitive response (HR) due to execution of localized cell death (Grant and Loake, 2000). ROS target polyunsaturated lipids such as linoleic acid (18:2) and αlinolenic acid (18:3) resulting in accumulation of highly reactive α,βunsaturated carbonyl compounds (Reactive Electrophilic Species; RES),

Corresponding author at: Department of Plant Pathology, University of Florida, 1453 Fifield Hall, Gainesville, FL 32611, USA. E-mail address: dgabr@ufl.edu (D.W. Gabriel).

https://doi.org/10.1016/j.jplph.2019.03.001 Received 10 December 2018; Received in revised form 8 February 2019; Accepted 1 March 2019 Available online 05 March 2019 0176-1617/ © 2019 Elsevier GmbH. All rights reserved.

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BM7 medium and a 10 μl cell suspension from each dilution was spotted in triplicate on BM7 agar plates. The plates were incubated at 28 °C for three weeks and colony counts (CFU) were enumerated. Agrobacterium-infiltrated, tBOOH-treated (0 and 50 mM) citrus leaf discs were ground in cold mortars and pestles in lysis buffer RLT (Qiagen RNeasy Plant Mini Kit; Qiagen, Valencia, CA, USA). RNA was extracted following the manufacturer’s protocol, diluted with nuclease free water to 500 ng μl−1 and cleaned with TURBO DNA-free (DNase) Kit (Ambion, Austin, TX, USA). Leaf discs treated with 75 mM tBOOH were too damaged to yield good quality RNA for transcript analyses. Reverse transcription (RT) reactions were performed using one μg RNA template (iScript Advanced cDNA Synthesis Kit; Bio-Rad, Hercules, CA, USA). Quantitative RT-PCR analyses were performed using a CFX96 Touch Real-Time PCR detection system (Bio-Rad) as described (Jain et al., 2015). All primers used are summarized in Supp. Table S1 and the gene expression data were normalized against elongation factor 1-α (CitEF1α). All data were analyzed using SAS for Windows, version 9.2 (SAS Institute Inc, Cary, NC) and Student's t tests were performed to separate treatment means. A probability of 5% was used to determine statistically significant differences.

primarily oxylipins. Oxylipins are a diverse class of oxygenated unsaturated fatty acid derivatives with well known signaling functions in plants and animals (Farmer and Müller, 2013). Oxylipins formed in plants include jasmonates, divinylethers and green leaf volatiles (GLVs). Jasmonates include 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA) and methyl jasmonate, and GLVs consist of C6 and C9 aldehydes, alcohols and their esters (ul Hassan et al., 2015). Both ROS-mediated lipid peroxidation as well as dioxygenation of fatty acids by lipoxygenases (LOXs, EC 1.13.11.12), form the initial committed steps in oxylipin biosynthesis (Farmer and Müller, 2013). The basal levels of oxylipins derived from ROS-mediated lipid peroxidation are generally in the same range or even above those of the products of enzymatic peroxidation (Müller, 2004). Oxylipins contribute to plant defense by direct antimicrobial activity, transcriptional activation of defense genes and regulation of HR-mediated localized cell death (Prost et al., 2005). Peroxiredoxins (EC 1.11.1.15) are a ubiquitous and diverse family of thioredoxin-scaffold enzymes with cysteine-dependent peroxidase activity against hydrogen peroxide, peroxynitrite and organic peroxide substrates. Peroxiredoxins utilize either 2-Cys or 1-Cys reaction catalysis mechanisms, the latter of which is redox insensitive (Nelson et al., 2011). 2-Cys peroxiredoxins play an additional important role in H2O2 accumulation and signaling in eukaryotes (Nelson et al., 2011). In Las, CLIBASIA_RS00445 encoded LasBCP (WP_012778432.1) was recently demonstrated to be an extracellular 1-Cys peroxiredoxin essential for survival and productive colonization in planta. LasBCP was annotated as an H2O2 degrading enzyme with substrate specificity for organic peroxides and was functionally confirmed to suppress RBOH-mediated long distance systemic signaling and accumulation of oxylipins in planta (Jain et al., 2018). In the present report we provide further evidence that, (a) oxylipins generated by citrus inhibited growth of nonpathogenic L. crescens strain BT-1 (Leonard et al., 2012), a cultured surrogate for the uncultured pathogenic Liberibacters; and (b) transient expression of LasBCP in citrus leaves attenuated JA and salicylic acid (SA) defense signaling pathways.

3. Results 3.1. LasBCP alleviated tBOOH-induced lipid peroxidation in citrus leaves Leaf disc senescence assays were used to quantify total chlorophyll content as a visual indicator of tBOOH-induced oxidative damage in citrus leaf discs following Agrobacterium-mediated transient expression of GUS or LasBCP from the binary plasmids pCAMBIA2301 (CaMV35S::uidA) and pAMB004 (CaMV35S::Lasbcp), respectively (Fig. 1A). As in previously published studies (Jain et al., 2015, 2018), maximum expression of both GUS and LasBCP was confirmed by RTPCR at 4 days post infection (not shown). Exogenously applied tBOOH (25, 50 and 75 mM) caused dose-dependent bleaching and significant declines in chlorophyll content in GUS-expressing leaf discs (30, 41 and 70%, respectively) as compared to untreated leaves. In comparison, leaves expressing LasBCP exhibited significantly less chlorophyll damage (19, 32 and 47%, respectively) as compared to the controls under similar tBOOH treatment conditions (Fig. 1B). Oxidative damage to free and membrane-bound lipids was quantified by determining the total malondialdehyde (MDA, an oxylipin) content in Agroinfiltrated citrus leaf discs challenged with 25, 50 and 75 mM tBOOH (Fig. 1C). Similar MDA levels were observed in untreated leaf discs expressing either GUS or LasBCP (2.03 ± 0.13 and 1.98 ± 0.16 nmole MDA μg−1 fresh weight, respectively). Consistent with the chlorophyll loss data, tBOOH (25, 50 and 75 mM) treatments resulted in increased MDA levels in citrus leaf tissues expressing GUS (2.3, 2.7 and 4.6-fold, respectively). By contrast, LasBCP expression in citrus leaf discs provided considerable protection against lipid peroxidation, resulting in significantly lower 1.4, 1.9 and 3.1-fold increases in MDA content when exposed to 25, 50 or 75 mM tBOOH, respectively. These results indicated a determinative role of LasBCP in mitigating lipid peroxidation-mediated damage in Las infected in citrus leaves.

2. Materials and methods Lasbcp was PCR-amplified from DNA extracts of Las-infected citrus and cloned in pCR2.1-TOPO (Invitrogen, Carlsbad, CA, USA) (Jain et al., 2018). Following sequence verification, Lasbcp was directionally subcloned into pCAMBIA2301 (CaMV35S::uidA) (Hajdukiewicz et al., 1994) replacing uidA. The resulting plasmid pAMB004 (CaMV35S::Lasbcp) was mobilized into A. tumefaciens strain GV2260 via electroporation. Agrobacterium-mediated transient expression assays in citrus were performed as described (Hao et al., 2014). Four days-postinfiltration Agrobacterium-infiltrated citrus leaf discs were floated on liquid Murashige and Skoog (MS) basal medium (Murashige and Skoog, 1962) containing 0–75 mM tert-butyl hydroperoxide (tBOOH, an organic peroxide) for two days. Total chlorophyll pigments in the leaf discs were extracted overnight in cold 80% (v/v) acetone and quantified according to the procedure of Arnon (1949). Malondialdehyde (MDA), the lipid peroxidation reaction endproduct was measured using the MDA assay kit (Sigma-Aldrich, St. Louis, MO, USA). MDA reacts with thiobarbituric acid to form a fluorimetric product (λex/em = 532/553 nm). L. crescens BT-1 cells were maintained on BM7 medium with gentle shaking at 150 rpm at 28 °C as described (Jain et al., 2015). Crude aqueous cell-free extracts of Agrobacterium-infiltrated and tBOOHtreated citrus leaf discs were prepared by pulverizing the leaf tissue to a fine powder under liquid nitrogen and resuspending in BM7 medium (500 mg ml−1). The aqueous extracts were cleared by centrifugation twice at 3220 × g for 20 min at 4 °C and filter sterilized. Five-day-old L. crescens BT-1 cells were harvested and resuspended in leaf MDA extract/BM7 medium to a density of Abs600 = 0.5 and incubated for 12 h. For the cell viability assays, a 10-fold serial dilution was prepared in

3.2. Citrus leaf oxylipins inhibited growth of Liberibacter crescens in culture In a conventional spot titration assay (Fig. 2), L. crescens BT-1 cells were found to be highly sensitive to 50 nmole MDA (1.8 × 104 CFU ml−1) and oxylipins extracted from GUS-expressing citrus leaves treated with 50 mM tBOOH (5.4 × 105 CFU ml−1) by comparison with the untreated BT-1 cells (3.2 × 107 CFU ml−1). Extracts from LasBCP expressing leaf tissue treated with 50 mM tBOOH clearly exhibited significantly less antimicrobial activity to BT-1 cells (8.6 × 106 CFU ml−1) than extracts from similarly treated GUS expressing citrus leaf tissue, presumably due to suppressed biosynthesis and accumulation of 62

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Fig. 2. Candidatus Liberibacter asiaticus peroxiredoxin LasBCP attenuated accumulation of antimicrobial oxylipins in citrus leaves. Leaf discs transiently expressing GUS or LasBCP from the binary plasmids pCAMBIA2301 (CaMV35S::uidA) or pAMB004 (CaMV35S::Lasbcp) were floated for two days on liquid MS medium containing 50 mM tBOOH, and used for oxylipin extraction. Five-day-old L. crescens BT-1 cells were harvested, resuspended in leaf oxylipin extracted in BM7 medium (Abs600 = 0.5) for 12 h, washed, serially diluted and plated on BM7 media. The surviving bacterial colonies were scored after three weeks. The data presented are the means ± SE for three independent experiments with two replicates. Means ( ± SD) marked with different letters are significantly different based on Tukey-Kramer’s mean separation (P ≤ 0.05).

(CitJAZ1) (P = 0.002254) was upregulated in LasBCP-expressing citrus leaves compared to GUS controls. The key SA biosynthesis and SAR genes phenylalanine ammonia lyase (CitPAL) (P = 0.002584), isochorismate synthase (CitICS) (P = 0.000206) and pathogenesis related protein-1 (CitPR1) (P = 0.000001) were also transcriptionally downregulated in LasBCP-expressing citrus leaves compared to the GUS-expressing leaf tissue. In similar experiments, expression of two endogenous housekeeping genes cytosolic glyceraldehyde-3-phosphate dehydrogenase (CitGAPDH) and SAND family protein (CitSAND; required for vesicular traffic) remained unchanged. The transcript normalization calibrator gene CitEF1α, as well as CitGAPDH and CitSAND have previously exhibited reproducible transcriptional stability in different citrus organs under biotic stress (Killiny and Nehela, 2017). In the absence of tBOOH-induced peroxide damage, transcript levels of JA and SA defense pathway genes were not significantly affected between GUS- and LasBCP-expressing leaf tissue (data not shown).

Fig. 1. Candidatus Liberibacter asiaticus peroxiredoxin LasBCP alleviated oxidative damage to membrane lipids in citrus leaves. (A) Leaf discs transiently expressing GUS or LasBCP from the binary plasmids pCAMBIA2301 (CaMV35S::uidA) or pAMB004 (CaMV35S::Lasbcp) were floated for two days on liquid MS medium containing 0–75 mM tBOOH as indicated, and harvested for quantification of (B) total chlorophyll (μg mg−1 fresh weight) and (C) MDA (nmole μg−1 fresh weight) content. The data represent average of three independent experiments with four replicates. Means ( ± SD) marked with different letters are significantly different based on Tukey-Kramer’s mean separation (P ≤ 0.05).

oxylipins.

4. Discussion

3.3. LasBCP suppressed transcriptional activation of defense signaling pathway genes in citrus leaves

Plants respond to an attempted pathogen invasion via an apoplastic oxidative burst (H2O2) resulting in localized accumulation of lipid radicals and subsequent biosyntheses of a diverse array of oxygenated unsaturated fatty acid derivatives collectively termed oxylipins. Transient expression of LasBCP in citrus leaves provided significant resistance to exogenously applied organic peroxide tBOOH, thereby protecting free and membrane-bound lipids against peroxidative damage and preserving the molecular integrity of the chlorophyll apparatus (Fig. 1). L. crescens viability in culture was significantly reduced when exposed to lipid peroxidation products (MDA and other oxylipins) accumulated in tBOOH-treated citrus leaves expressing GUS (Fig. 2). However, a similar oxylipin extract obtained from LasBCP-expressing leaves was less inhibitory to L. crescens growth and viability. These data clearly demonstrated a predicted protective role of secreted LasBCP in favor of Liberibacter survival and colonization by alleviating ROS-induced lipid peroxidation in citrus, a host for Las. H2O2-mediated accumulation of antimicrobial oxylipins is believed to be a pivotal and

Expression levels of several key marker genes of the JA and SA defense signaling networks were examined in citrus leaves following transient expression of GUS (control) and LasBCP with or without treatment with 50 mM tBOOH for two days (Fig. 3). The JA biosynthesis genes ω-3 FA desaturase (CitFAD) (P = 0.001326), linoleate lipoxygenase (CitLOX) (P = 0.002674), allene oxide synthase (CitAOS) (P = 0.001923) and allene oxide cyclase (CitAOC) (P = 0.005776) were significantly downregulated in LasBCP expressing citrus leaves compared to GUS controls, suggesting lipid peroxidation-mediated downregulation of oxylipin accumulation (refer Fig. 1B). Also, in LasBCPexpressing citrus leaves, expression levels of the key JA signaling genes jasmonoyl isoleucine synthetase (JASMONATE RESISTANT 1, CitJAR1) (P = 0.001230), CORONATINE-INSENSITIVE 1 (CitCOI1) (P = 0.000004) and JASMONATE INSENSITIVE 1 (CitJIN1, also known as Myc2) (P = 0.001233) were significantly downregulated. However, the negative regulator of JA response genes JASMONATE ZIM DOMAIN 63

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In addition to the oxylipin biosynthetic genes, JA signal transduction (Wasternack and Strnad, 2016) genes encoding JAR1, COI1 and JIN1 were all similarly suppressed in a coordinated manner by LasBCP, whereas JAZ1, a negative regulator of JA signal transduction, was transcriptionally upregulated (Fig. 3). Amide-linked conjugation of JA with Ile [(+)-7-iso-jasmonoyl-L-isoleucine, JA-Ile] via JAR1 activity is essential for physiological JA function. Downstream transduction of bioactive JA-Ile conjugates depends on COI1, which mediates E3 ubiquitin ligase activity of the 26S proteasome (Skp1/Cullin/F-box or SCFCOI1) complex. Degradation of transcription repressor JAZ1 by the 26S proteasome relieves repression of JIN1-upregulated JA responsive genes. Killiny and Nehela (2017) reported an increase in JA content only in response to D. citri herbivory but not following Las-infected D. citri attack. JA-Ile conjugates are required for executing effective defense against Las. The Ile content is significantly lower in the phloem sap of Las-susceptible citrus varieties (Killiny and Hijaz, 2016). It is conceivable that reduced Ile further compromises availability of JA-Ile conjugates required for executing effective defense against Las. In addition, the Ile content is further reduced in symptomatic Las-infected leaves (Slisz et al., 2012). Las cannot synthesize Ile and is reliant upon its host to provide Ile. It is also interesting to note that Las incorporates significantly more Ile in its proteins than other free-living Rhizobiales such as Agrobacterium (Hartung et al., 2011), providing an Ile sink that may also contribute to reduced JA signaling within the infected host. Activation of SAR in response to localized biotrophic microbial infections and the subsequent HR is dependent upon endogenous accumulation of SA and activation of a battery of pathogenesis-related (PR) genes. The NON-EXPRESSOR OF PATHOGENESIS-RELATED (PR)1 (NPR1) gene mediates the reciprocal inhibition of JA responses (to necrotrophy and herbivory) by the SA signaling pathway (Gfeller et al., 2006). However, several studies have also provided evidence contrary to antagonistic SA/JA pathways, indicating overlapping and synergistic roles for both hormones in ROS generation and induction of SAR (AmilRuiz et al., 2016; Zhu et al., 2014; Truman et al., 2007). The concurrent suppression of JA and SA biosynthetic and signaling pathway genes by LasBCP in citrus leaves in response to lipid peroxidation (Fig. 3) supports a synergistic role for of JA and SA in citrus defense against Las. Phloem sap and sieve tube elements serve as checkpoints for the generation and transport of H2O2 required for the systemic immune signaling cascade (Kadota et al., 2015; Van Bel and Gaupels, 2004). Ample evidence exists to show that JA biosynthetic enzymes are localized in the sieve tube elements and companion cells (Hause et al., 2003), consistent with the role of oxylipins as mobile regulatory components of distally activated immune responses. We hypothesize that extracellular LasBCP simultaneously alleviates (a) auto-propagation of the RBOH-generated H2O2 feedback loop, (b) H2O2-mediated peroxidation of free and membrane lipids, and (c) the consequent increase in antimicrobial and regulatory oxylipin burden (Figs. 1–3; Jain et al., 2018). LasBCP, which is expressed in citrus but not in psyllids (Jain et al., 2018), thereby inhibits both long-distance innate immunity as well as ROS-mediated localized hypersensitive cell death in phloem sieve tube elements. This activity likely enables phloem-limited Las to evade early detection and clearing in citrus. LasBCP is a promising target for chemical inactivation and development of chemical control methods for HLB, at least in early stages of citrus infection.

Fig. 3. Candidatus Liberibacter asiaticus peroxiredoxin LasBCP suppressed jasmonic acid and salicylic acid defense signaling genes in citrus leaves. Expression levels of jasmonic acid (CitFAD, CitLOX, CitAOS, CitAOC, CitJAR1, CitCOI1, CitJIN1, CitJAZ1) and salicylic acid (CitPAL, CitICS, CitPR1) biosynthetic and signaling genes, and two housekeeping genes (CitGAPDH and CitSAND) were compared in leaf discs transiently expressing GUS or LasBCP from the binary plasmids pCAMBIA2301 (CaMV35S::uidA) or pAMB004 (CaMV35S::Lasbcp) and floated for two days on liquid MS medium containing 50 mM tBOOH. Transcript data were normalized against expression of elongation factor 1-α (CitEF1α). The data presented are the means ± SD for three independent experiments with four replicates. Asterisk indicates a significant difference between the treatment means (P ≤ 0.05).

relatively ancient host defense response, prevalent before the evolution of enzymatic oxylipin defense signaling cascades (Müller, 2004). Compelling evidence has demonstrated the role of JA and SA in activating plant immunity through recognized signaling pathways (Gfeller et al., 2006). Consistent with our data (Fig. 3), several lines of evidence indicate suppression of oxylipin-mediated defense signaling during Las infection. For instance, emission of GLVs was compromised in citrus following Las-infected psyllid attack (Hijaz et al., 2013) and highly Las-susceptible citrus cultivars contained relatively lower amounts of leaf volatiles than the more Las-resistant (HLB tolerant and moderately tolerant) varieties (Hijaz et al., 2016). GLVs prime defense reactions in plants by upregulating constitutive levels of free unsaturated fatty acids, resulting in stronger and faster JA-mediated defense signaling (Li et al., 2016). Targeted metabolomic analyses revealed significant reductions in long-chain fatty acid biosyntheses and their oxygenated fragmented products in Las-infected citrus (Suh et al., 2018). While the LOX levels were downregulated in Las-infected citrus leaves (Nwugo et al., 2016; Fan et al., 2011), the increase in LOX substrates in leaves caused by D. citri infestation was rapidly attenuated upon coinfection with Las (Killiny and Nehela, 2017). The coordinated transcriptional suppression of LOX, AOS and AOC in LasBCP-expressing citrus leaves suggested active downregulation of oxylipin, including JA, accumulation (Fig. 3).

Acknowledgements We thank Patricia Rayside for excellent technical assistance. This work was supported by the Florida Citrus Research and Development Foundation (CRDF) project #15-009 and by USDA-NIFA-SCRI grant #2016-70016-24844.

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