ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 3, с. 376-383
ЭКСПЕРИМЕНТАЛЬНЫЕ ^^^^^^^^^^^^ СТАТЬИ
УДК 581.1
Pst DC3000 INDUCES PATHOGENESIS-UNCORRELATED CYTOSOLIC
Ca2+ RISE IN Arabidopsis LEAVES1 © 2014 H. Sun2, C. Ma2, X. Ren, Y. Zhang, H. Wang, Y. He, A. Hasi, Y. Kang
College of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China
Received March 11, 2013
In nature, the phytopathogen usually initiates its infection on the leaf surface before moving into the internal space through natural openings. Little is known about immediate response of the leaf to the surface-colonizing phytopathogen and its correlation with individual microbe-associated molecular patterns (MAMPs). In this study, we monitored the dynamic changes in the cytosolic Ca2+ concentration ([Ca2+]cyt) in the Arabidopsis leaf expressing luminescence protein aequorin as the response to the surface-inoculating Pseudomonas syringae DC3000 (Pst DC3000) with a touching-free system. The significant [Ca2+]cyt transient rise was evoked in the leaf right after inoculation, and its magnitude was correlated with the pathogen concentration. Pharmacological studies revealed that the rising [Ca2+]cyt occurs primarily from the cAMP-mediated Ca2+ mobility pathway, but not Gd3+-sensitive Ca2+ influx channel in the plasma membrane, which was distinct from those induced by individual MAMPs (lipopolysaccharide, flagellin, and elongation factor Tu). Pretreat-ing the leaf with Pst DC3000 or MAMPs significantly attenuated its responses to subsequent treatments of any of them, which indicates that the leaf has the convergent mechanism of sensitivity to the pathogen and MAMPs. Furthermore, Pst DC3000 mutants defective in flagellum, type III secretion apparatus, and phyto-toxin coronine production significantly lost their multiplication ability in the leaf apoplast, but evoked [Ca ]
cyt responses comparable with that of the wild type. Taken together, these data indicates that the [Ca2+]cyt in the leaf has the sensitive response to the surface-inoculating phytopathogen, which was distinct from those of individual MAMPs and had no correlation with the pathogen pathogenesis capacity.
Keywords: Arabidopsis — Ca2+ — transient rise — Pst DC3000 — MAMPs
DOI: 10.7868/S0015330314030130
INTRODUCTION
Current knowledge about molecular mechanism of the bacterial pathogen-plant interaction is built primarily on the research with the model pathogen Pseudomonas syringae DC3000 (Pst DC3000) and plant Arabidopsis thaliana [1]. Pst DC3000 is a gramnegative plant pathogen, which elicits a wide variety of innate immunity responses in plant cells through its unique MAMPs being recognized by corresponding receptors in the plasma membrane [2]. MAMPs of Pst DC3000 include lipopolysaccharides (LPS), major component of the outer membrane; flagellin, and elongation factor Tu (EF-Tu), the most abundant bac-
1 This text was submitted by the authors in English.
2 These authors contributed equally to this study.
Abbreviations'. AP-5 - 2-amino-5-phosphonopentanoic acid; CFU - colony-forming unit; DND1 - defense no death; EFR -elongation factor receptor; FLS2 - flagellin-insensitive 2; GLR -glutamate receptor; HR - hypersensitive response; LPS - lipopolysaccharides; MAMPs - microbe-associated molecular patterns; Pst DC3000 - Pseudomonas syringae DC3000. Corresponding author. Yan Kang. College of Life Sciences, Inner Mongolia University, Hohhot 010021, Inner Mongolia, China. Fax. +86-471-499-2435; e-mail. kangyan105@imu.edu.cn
terial protein, or their peptide surrogates flg22 and elf18, respectively [3].
In the Arabidopsis cellular plasma membrane, there are flagellin-insensitive 2 (FLS2) and elongation factor receptor (EFR), specifically recognizing flg22 and elf18, respectively [4]. Receptor for LPS, which is recognized by Toll-Like Receptor 4 in animal cells with similar structure as the AtFLS2 and AtEFR, has not been identified yet in the plant cell [5]. The pathogen can deliver pathogenic protein effectors into contacting plant cells through its type III secretion system [6]. These effectors can disarm MAMPs-evoked innate immunity responses of susceptible plant cells [7] or are recognized by specific proteins in the plant cell to initiate hypersensitive response (HR) in the resistant plant cells [8]. HR is a form of a rapid localized programming cell death with mercy of saving the rest of the leaf from collapse [9].
Natural infection process of Pst DC3000 usually involves three steps: establishing epiphytic colonization; moving into natural openings, primary stomata or wounding sites; and entering the entophytic apo-plast space [2]. Innate immunity systems of plant epidermal, guard cells of the stomata, and the mesophyll
cells are sequentially challenged by the pathogen during each of these steps [10]. To mimic the natural process, two artificial pathogen inoculation procedures are developed: pathogen spraying on the leaf surface [11], dipping the leaves of the whole soil-grown plants into, or flooding the young seedlings with the pathogen suspension [10, 12], all of which involve innate immunity of the epidermal and guard cells; another way is to force pathogen going through natural openings on the leaf surface into the apoplast space through pressure-based infiltration, which essentially bypasses innate immunity of the epidermal and guard cells [13].
There are two typical cases highlighting the qualitative difference of immunity responses evoked by the two inoculation procedures. Firstly, Arabidopsis sto-mata closure is induced while the AtFLS2 in the guard cell plasma membrane senses the pathogen's flagellin, which keeps the pathogen from invading into the apo-plast [14]. Therefore, Arabidopsis fls2 null mutant is more susceptible than the wild type to the pathogens sprayed onto the leaf surface, but shows the wild-type phenotype at pathogen infiltration [11]. Secondly, non-protein virulence effector coronatine facilitates Pst DC3000 penetration through stomata by suppressing innate immunity response of the guard cells [14]. Therefore, Pst DC3000 defective in coronatine production does not cause significant disease symptoms when being sprayed onto the leaf surface, but does at infiltration. It implies that the innate immunity system of the epidermal and guard cells differs essentially from that of endophytic mesophyll cells.
Accumulating evidences obtained with various plant species and tissues implies an essential role of the plant [Ca2+]cyt in MAMP sensing and initiating subsequent defense responses [15]; the exact mechanism is far from clear. There are three studies specifically focused on the Pst DC3000 and Pst DC3000 avrRpm1 suspension-induced [Ca2+]cyt transient rise in Arabi-dopsis mesophyll cells after being infiltrated into the entophytic space under artificial pressure [16, 17]. The infiltration inevitably induces strong touching-evoked [Ca2+]cyt transient elevation. It came down to the resting level after 5 min and is followed by subsequent two [Ca2+]cyt transient rises, the first at 10 min and the second at 120 min [16, 18]. The first is inhibited by adenylate cyclase inhibitor alloxan and Ca2+ influx channel inhibitor GdCl3 [16]; the second is fully dependent on the interaction between the pathogen effector avrRpm1 and corresponding protein AtRPS3 in the plant as well as partially dependent on two calreticulin proteins, AtCRT1 and AtCRH1 [17, 18]. By far little is known about early signaling events in plant cells occurring right after bacterial pathogen arriving to the leaf surface and what is the correlation of these events, if any, with downstream pathogen infection capacity, as well as the correlation between the signaling cascades in plant cells induced by pathogen itself and individual MAMPs. In this study, Pst DC3000 suspen-
sion was delivered into a solution containing an Arabidopsis detached leaf expressing [Ca2+]cyt-dependent luminescence protein aequorin from jellyfish [19]. This system is completely free from artificial manipulation of the interaction, in which pathogens get contacts with the leaf surface through their own diffusion process. With this system, we demonstrated that (1) epiphytic inoculation of Pst DC3000 induces the pathogen concentration-dependent and a receptor-mediated heterogeneous process, which have both convergent and divergent signaling cascades from known MAMPs; (2) the response is not correlated with the pathogen infection capacity.
MATERIALS AND METHODS
Plant materials and bacterial preparation. Arabidopsis wild type Columbia-0 and dnd1 mutant expressing cytosolic Ca2+-dependent luminescence protein aequorin were described previously [16, 19]. Arabidopsis seeds were surface-sterilized with 75% ethanol for 10 min and 100% ethanol for 5 min and then dried on sterilized filter paper. The 10 seeds were sown evenly on growth medium containing half-strength MS basal salts, 1% sucrose, 5 mM Mes, and 0.7% agar, pH 5.7. Plates were placed horizontally in growth chamber for 18-21 days under 100 ^E/(m2 s) light intensity, a 12-h photoperiod, and 22-25°C.
Bacteria for treatment were directly streaked from -80°C freezer stock onto low salt Luria-Berani plates (tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L) with appropriate antibiotics: 50 ^g/mL Rifampin for Pst DC3000; 50 ^g/mL Rifampin, 35 ^g/mL chloram-phenicol for Pst DC3000 defective with flagellin (Pst DC3000fa"); 50 ^g/mL Rifampin, 25 ^g/mL kan-amycin for Pst DC3000 defective in gene HrpA for the type III secretion apparatus (Pst DC3000 hrpA-) and for Pst DC3000 defective in phytotoxin coronine (Pst DC3000 cor-). Plates with bacteria were cultured at 28°C for 38-42 h. Bacteria were scratched from the boundary of the streak into 1 mL of control buffer containing 1 mM KCl, 1 mM CaCl2, and 5 mM Mes, pH 5.7. Subsequently, they were vortexed and washed with the control buffer three times at 1000 g centrifug-ing. The final bacterial concentration was adjusted to OD600 of 2.
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