SlCNGC1 and SlCNGC14 Suppress Xanthomonas oryzae pv. oryzicola-Induced Hypersensitive Response and Non-host Resistance in Tomato

Mechanisms underlying plant non-host resistance to Xanthomonas oryzae pv. oryzicola (Xoc), the pathogen causing rice leaf streak disease, are largely unknown. Cyclic nucleotide-gated ion channels (CNGCs) are calcium-permeable channels that are involved in various biological processes including plant resistance. In this study, functions of two tomato CNGC genes SlCNGC1 and SlCNGC14 in non-host resistance to Xoc were analyzed. Silencing of SlCNGC1 and SlCNGC14 in tomato significantly enhanced Xoc-induced hypersensitive response (HR) and non-host resistance, demonstrating that both SlCNGC1 and SlCNGC14 negatively regulate non-host resistance related HR and non-host resistance to Xoc in tomato. Silencing of SlCNGC1 and SlCNGC14 strikingly increased Xoc-induced callose deposition and strongly promoted both Xoc-induced and flg22-elicited H2O2, indicating that these two SlCNGCs repress callose deposition and ROS accumulation to attenuate non-host resistance and PAMP-triggered immunity (PTI). Importantly, silencing of SlCNGC1 and SlCNGC14 apparently compromised cytosolic Ca2+ accumulation, implying that SlCNGC1 and SlCNGC14 function as Ca2+ channels and negatively regulate non-host resistance and PTI-related responses through modulating cytosolic Ca2+ accumulation. SlCNGC14 seemed to play a stronger regulatory role in the non-host resistance and PTI compared to SlCNGC1. Our results reveal the contribution of CNGCs and probably also Ca2+ signaling pathway to non-host resistance and PTI.


INTRODUCTION
Each pathogen has its own host range. Non-host resistance is triggered when a non-adapted pathogen attempts to infect a plant species outside of its host range. Thus, non-host resistance is widely occurring, durable and broad-spectrum to non-adapted pathogens and is highly potential to be exploited in crop resistance engineering (Schulze-Lefert and Panstruga, 2011;Senthil-Kumar and Mysore, 2013). It has been clear that plant non-host resistance utilizes both preformed and induced defense mechanisms and frequently elicites hypersensitive response (HR) (Mysore and Ryu, 2004;Senthil-Kumar and Mysore, 2013). The preformed mechanisms generally consist of plant physical and chemical barriers, including antibiotic compounds (Bednarek and Osbourn, 2009;Fan et al., 2011). The induced non-host resistance is elicited after the preformed defense is overcome. As observed for host resistance, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) are often initiated in this layer of defense (Niks and Marcel, 2009;Senthil-Kumar and Mysore, 2013). Efforts to identifiy non-host resistance-related genes resulted in the finding that some genes, such as PLDδ, GOX, SGT1, and NHO1, contribute to both host and non-host resistance in Arabidopsis (Lu et al., 2001;Maimbo et al., 2010;Rojas et al., 2012;Pinosa et al., 2013). Generally speaking, the mechanisms underlying plant non-host resistance are far from well understood.
Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) are two important pathovars of X. oryzae, causing bacterial blight and leaf streak diseases, respectively, in rice (Oryza sativa), which is a staple food in many countries and a model plant for cereal biology (Nino-Liu et al., 2006). It is notable that these two pathovars utilize distinct mechanisms to infect rice leaves. Xoo is a vascular pathogen that enters rice leaves via the hydathodes, while Xoc penetrates rice leaves mainly through stomata or wound sites and colonizes intercellular spaces in the mesophyll (Nino-Liu et al., 2006). During infection of their plant hosts, many strains secrete transcription activator-like (TAL) effectors, which enter the host cell nucleus and activate specific corresponding host genes at effector binding elements (EBEs) in the promoter (Boch et al., 2009;Moscou and Bogdanove, 2009). It has been established that host resistance to Xoo is mostly related to the action of TAL effectors, either by polymorphisms that prevent the induction of susceptibility (S) genes or by executor resistance (R) genes with EBEs embedded in their promoter, and that induce cell death and resistance (Bogdanove et al., 2010;Zhang et al., 2015). Xoc is known to suppress host resistance, and no host R gene has been identified against it (Cai et al., 2017). Compared with the host resistance (Zhang and Wang, 2013), non-host resistance to Xoo and Xoc is much less studied. We have previously identified seven genes required for non-host resistance to Xoo in Nicotiana benthamiana. Among them are a calreticulin and a peroxidase, indicating that oxidative burst and calcium-dependent signaling pathways play an important role in non-host resistance to Xoo (Li et al., 2012). However, molecular mechanisms underlying non-host resistance to Xoc remain largely unknown. Whether the oxidative burst and calcium signaling pathway contribute to the non-host resistance to Xoc as to Xoo awaits examination.
The cyclic nucleotide-gated ion channels (CNGCs) are suggested to be one of the important pathways for conducting Ca 2+ ions in signaling transduction (Talke et al., 2003). They are ligand-gated Ca 2+ -permeable divalent cation-selective channels that are often localized in plasma membrane, presumptively are activated by direct binding of cyclic nucleotides and are complexly regulated by binding of calmodulin (CaM) to the CaM Binding (CaMB) domain (Chin et al., 2009;Ma et al., 2009;Wang et al., 2013;Gao et al., 2014;DeFalco et al., 2016a,b;Fischer et al., 2017). The plant CNGCs are involved in numerous biological functions varying from plant development and stress tolerance to disease resistance (Kaplan et al., 2007;Qi et al., 2010;Ma and Berkowitz, 2011;Moeder et al., 2011). CNGCs are well conserved among plant species, comprising 20 members in Arabidopsis and 18 members in tomato (Saand et al., 2015a,b). Earlier studies revealed that AtCNGC2, AtCNGC4, AtCNGC11, and AtCNGC12 and their homologs play an important role in disease resistance against various pathogens (Yu et al., 1998;Clough et al., 2000;Balagué et al., 2003;Jurkowski et al., 2004;Yoshioka et al., 2006;Ali et al., 2007;Ma and Berkowitz, 2011;Chin et al., 2013;Fortuna et al., 2015;Saand et al., 2015a). Later, other CNGC members such as SlCNGC1 and SlCNGC14 were also found to contribute to disease resistance (Saand et al., 2015a,b). We found that SlCNGC1 and SlCNGC14 play a negative role in non-host resistance to Xoo in tomato (Saand et al., 2015b). However, whether these SlCNGCs indeed encode functional CNGC channels and whether and how they function in non-host resistance to Xoc in tomato remains further study.
Our data in this study strongly indicate that SlCNGC1 and SlCNGC14 function as Ca 2+ channels and negatively regulate tomato non-host resistance to Xoc through modulating ROS accumulation and callose deposition. Our results provide evidence for the contribution of CNGC-mediated Ca 2+ signaling pathway to non-host resistance.

Plant Growth and Inoculation
Tomato (cv. Money Maker) plants were grown in growth room at 21 • C with16 h light/8 h dark photoperiod. For disease resistance evaluation analyses, tomato plants were inoculated with X. oryzae pv. oryzicola (Xoc). After single colony propagation culture in NA medium, the Xoc bacterial cells were collected and resuspended in ddH 2 O to 1 × 10 8 cfu mL −1 . The bacterial suspension was then infiltrated into leaves with sterilized needleless syringe. The infiltration zones were marked immediately after infiltration. The inoculated plants were grown in growth room at 26 • C with16 h light/8 h dark photoperiod.

Gene Silencing Analyses
The virus-induced gene silencing (VIGS) target fragments of SlCNGC1 (Solyc01g095770.2) and SlCNGC14 (Solyc03g114110.2) were amplified by RT-PCR with gene-specific primers (Supplementary Table S1) and ligated into the TRV-based VIGS vector pYL156, which was subsequently electroporated into Agrobacterium tumefaciens strain GV3101 for VIGS analyses. VIGS analyses were conducted with vacuum infiltration delivery approach as described using recombinant pYL156 with insertion of an eGFP fragment instead of an empty pYL156 as control to alleviate viral symptom (Saand et al., 2015a). At about 3 weeks post agro-infiltration, plants were inoculated with Xoc as described above.

Detection of Callose Deposition
Callose deposition were stained with aniline blue and visualized in fluorescence microscope. Briefly, the collected leaves were washed twice with ddH 2 O and ethanol, respectively, and cleared in acetic acid-ethanol (1:3) for 4 h. After washed twice with ddH2O, the leaves were incubated in aniline blue solution [150 mM KH 2 PO 4 , 0.1% (w/v) aniline blue, pH 9.5] for 1 h. The stained leaves were washed with ddH 2 O and observed in 30% glycerol by fluorescence microscopy.

Bacterial Number Counting Assays
Bacterial numbers in Xoc-infiltrated leaves of silenced plants were determined as previously reported (Li et al., 2012).

Detection of ROS
Xoc-inoculated leaves of tomato plants were detached and stained with 3,3-diamino benzidine hydrochloride (DAB) (1mg/mL) as described . Quantitative determination of PAMP-elicited H 2 O 2 was conducted using a luminol-based approach (Saand et al., 2015a). For each experiment, six tomato leaf disks of 3 mm at diameter from three plants were dipped in distilled water in the light over night. The leaf disks were then transferred into 50 µL of distilled water in a 96-well plate. After addion of the same volume mixture which contains 200 nM luminol (Sigma-Aldrich), 20 µg of horseradish peroxidase and 200 nM flg22, H 2 O 2 were measured for 35 min as luminescence using a Microplate Luminometer (Titertek Berthold, Germany).

Calcium Assay
Transient increase of cytosolic Ca 2+ concentration was monitored in the Aequarin transgenic tomato lines. For each experiment, six leaf disks were punched from three plants and vacuum-infiltrated in 12.5 µM coelenterazin h (Sigma) on a 96-well plate for 1 min and then set for 2 h at room temperature. Before measurement, the coelenterazin solution was gently removed and 200 µL of PAMP solution (100 nM flg22) was added to the wells. Luminescence was measured using a Microplate Luminometer (Titertek Berthold, Germany).

Gene Expression Analyses by qRT-PCR
Five defense-related Ca 2+ signaling genes SlCDPK10 (Solyc11g018610.1), SlCAMTA3 (Solyc04g056270.2), SlCBP60G (Solyc01g100240.2), SlCAM2 (Solyc10g081170.1) and SlCAM6 (Solyc03g098050.2) were subjected to gene expression analyses Rahman et al., 2016b;Wang et al., 2016). Total RNA was isolated by Trizol (TaKaRa, Japan) extraction according to the manufacture's instructions. RNAs were treated with DNase I (TaKaRa, Japan) and then reverse transcribed. Quantitative real time PCR (qRT-PCR) was performed as previously described using the StepOne Real-Time PCR system (Applied Biosystems, United States) with SYBR Green PCR Master Mix (TaKaRa, Japan) (Saand et al., 2015a). 18s rDNA was used as internal control. The accession number and the specific primers for amplification of the analyzed genes were listed (Supplementary Table S1). The expression of target genes relative to control was calculated based on a value of 2 − Ct as recommended by the manufacturer. Meanwhile, semi-qRT-PCR for these genes were conducted in parallel and obtained PCR products were analyzed by agarose gel electrophoresis.

Statistical Analyses of Data
All experiments were conducted three times independently. The quantitative measurement data were statistically analyzed using SPSS software and represent means ± standard error. Significant difference between mean values was determined with DMRT (p < 0.05).

Silencing of SlCNGC1 and SlCNGC14
Enhanced Xoc-Induced Hypersensitive Response and Non-host Resistance In order to dissect the function of SlCNGC1 and SlCNGC14 in Xoc-induced HR and non-host resistance, we used TRV-based VIGS vector to perform VIGS analyses for these genes. Transcripts of SlCNGC1 and SlCNGC14 genes accumulated to only about 20% of the eGFP silencing control ( Figure 1A), indicating that SlCNGC1 and SlCNGC14 genes had been effictiently silenced in these plants. Inoculation assays with Xoc in these silencing plants demonstrated that SlCNGC1-and SlCNGC14-silenced leaves showed more severe Xoc-induced HR than the eGFP control leaves. SlCNGC1-and SlCNGC14-silenced leaves showed obvious HR necrosis at 3 h post Xoc inoculation, and exhibited strong HR necrosis at 9 hpi, especially SlCNGC14-silenced leaves, which had turned into desiccative, while the eGFP control plants displayed barely visible and weak HR necrosis at 3 and 9 hpi, respectively ( Figure 1B). This result indicated that silencing of SlCNGC1 and SlCNGC14 enhanced Xoc-induced HR.
We further counted Xoc bacterial number in the infiltrated leaf areas. The result showed that compared with the eGFP controls, Xoc bacterial number in SlCNGC1-and SlCNGC14-silenced plants decreased significantly by 0.7 orders of magnitude at 9 hpi ( Figure 1C), indicating that silencing of SlCNGC1 and SlCNGC14 enhanced non-host resistance to Xoc.
Collectively, these results implied that both SlCNGC1 and SlCNGC14 might negatively regulate non-host resistance related HR cell death and non-host resistance to Xoc in tomato.

Silencing of SlCNGC1 and SlCNGC14 Increased Xoc-Induced Callose Deposition
To probe the mechanisms underlying SlCNGC1-and SlCNGC14dependent regulation of non-host resistance against Xoc, effect of these CNGC genes on callose deposition upon pathogen infection was examined. Callose deposition was visible in the aniline blue-stained leaves under the fluorescence microscopy. Microscopic observation result showed that SlCNGC1-and SlCNGC14-silenced leaves deposited much more callose than eGFP control leaves (Figure 2A). Further quantification revealed that callose deposits in SlCNGC1-and SlCNGC14-silenced leaves was 5.9-and 7.3-fold respectively, as many as in the eGFP control leaves at 4 hpi ( Figure 2B). This result demonstrates that SlCNGC1 and SlCNGC14 repress the callose deposition upon (C) Xoc bacterial number counting. Xoc bacterial numbers were counted in leaf areas that were inoculated with bacterium at 3 and 9 hpi. At least five leaves were tested for each treatment, and the data represent means ± standard error (SE). Significant differences between treatments and the control are indicated by an asterisk (p < 0.05).
pathogen infection to alleviate non-host resistance to Xoc in tomato.

Silencing of SlCNGC1 and SlCNGC14
Promoted ROS Accumulation Reactive oxygen species (ROS) is indispensable to X. oryzae pv. oryzae (Xoo)-induced HR and non-host resistance . Thus, we analyzed the effect of SlCNGC1 and SlCNGC14 on the production of hydrogen peroxide (H 2 O 2 ), one of the primary species of ROS. DAB staining result demonstrated that infiltration areas of SlCNGC1-and SlCNGC14-silenced leaves showed significantly stronger DAB staining than those of control ( Figure 3A), implying that silencing of SlCNGC1 and SlCNGC14 increased the production of H 2 O 2 .
Effect of SlCNGC1 and SlCNGC14 on the PAMP (flg22)-elicited H 2 O 2 accumulation was further examined using leaf disk assays and was indicated as relative luminescence (RLU). The luminol-based assay showed that flg22-induced H 2 O 2 in SlCNGC1-and SlCNGC14-silenced leaves culminated to over 4100 RLU, while that in eGFP control leaves was peaked only to 940 RLU (Figure 3B), demonstrating that silencing of SlCNGC1 and SlCNGC14 promoted the production of flg22-elicited H 2 O 2 .
Together, these results suggest that SlCNGC1 and SlCNGC14 supress the ROS accumulation to attenuate non-host resistance and PTI.

Silencing of SlCNGC1 and SlCNGC14
Compromised Cytosol Ca 2+ Influx Some Arabidopsis CNGCs have been proved to be functional Ca 2+ channels (Gao et al., 2014;Wang et al., 2017). To examine whether SlCNGC1 and SlCNGC14 function as Ca 2+ channels, FIGURE 2 | Silencing of SlCNGC1 and SlCNGC14 increased Xoc-induced callose deposition. Leaves inoculated with Xoc at 4 hpi were stained with aniline blue and visualized in fluorescence microscope. A typical staining result (A) and its quantification result (B) were shown. Significant differences between treatments and the control are indicated by an asterisk (p < 0.05). effect of silencing of these genes on accumulation of cytosolic Ca 2+ elicited by the PAMP flg22 was monitored through leaf disk assays using aequorin transgenic tomato lines. In eGFP control aequorin transgenic plants, flg22-triggered Ca 2+ increased rapidly and peaked to 192 RLU, while those in SlCNGC1and SlCNGC14-silenced aequorin transgenic plants strongly decreased with the peak value drop to only 97 and 39 RLU, respectively (Figure 4). This result indicates that SlCNGC1 and SlCNGC14 function as Ca 2+ channels and negatively regulate non-host resistance and PTI through modulating cytosolic Ca 2+ accumulation.

Silencing of SlCNGC1 and SlCNGC14 Altered Expression of Defense-Related Ca 2+ Signaling Genes
To obtain a clue to understand how SlCNGC1 and SlCNGC14 genes, as Ca 2+ channel genes, regulate plant disease resistance, we examined effect of silencing of these genes on expression of a set of defense-related Ca 2+ signaling genes to probe the possibility of their involvement in SlCNGC1-and SlCNGC14-mediated resistance regulation. The genes under this expression analysis included two CaM genes SlCaM2 and SlCaM6, a tomato homolog (SlCDPK10) of Arabidopsis calcium-dependent protein kinase gene AtCDPK11, and the tomato homologs (SlCBP60g and SlCAMTA3) of two Arabidopsis CaM-binding transcription factors AtCBP60g and AtCAMTA3. All these genes play an important role in regulating disease resistance (Du et al., 2009;Wang et al., 2009Wang et al., , 2011Boudsocq et al., 2010;Zhao et al., 2013;Sun et al., 2015;Rahman et al., 2016a,b;Wang et al., 2016). Result of qRT-PCR showed that at 4 h after inoculation with Xoc, SlCNGC1-and SlCNGC14-silenced leaves reduced expression of the negative defense regulatory gene SlCAMTA3 by 2.5-fold, and generally increased expression of the positive defense regulatory genes SlCaM6, SlCDPK10, and SlCBP60g to different extent, which was much higher in SlCNGC14-silenced leaves than SlCNGC1-silenced leaves. Expression of SlCaM6, SlCDPK10 and SlCBP60g genes in SlCNGC1-silenced leaves was 4.3-, 1.5-and 2.5-fold, respectively, as high as that in the eGFP controls, while these folds were 34.0, FIGURE 5 | Silencing of SlCNGC1 and SlCNGC14 altered expression of defense-related Ca 2+ signaling genes. Expression of defense-related Ca 2+ signaling genes SlCAM2, SlCAM6, SlCDPK10, SlCAMTA3, and SlCBP60g were examined for SlCNGC1-, SlCNGC14-silenced plants relative to the eGFP non-silenced control plants after inoculation with Xoc at 4 hpi by qRT-PCR with 18s rDNA gene serving as a loading control gene. Data represent the mean ± SE of three independent experiments. 4.5, and 9.1 for that in SlCNGC14-silenced leaves (Figure 5). This result implies that SlCNGC1 and SlCNGC14 might modulate the expression of these Ca 2+ signaling genes.

DISCUSSION
We previously found that two tomato CNGC genes SlCNGC1 and SlCNGC14 play an important role in tomato HR elicited by the rice bacterial blight pathogen X. oryzae pv. oryzae (Xoo) (Saand et al., 2015a). Here, we further reveal that these two CNGC genes likely encode functional Ca 2+ channels and negatively affect tomato non-host resistance to the rice bacterial leaf streak pathogen Xoc, through modulating ROS accumulation and callose deposition. The role of Ca 2+ signaling pathway in non-host resistance to Xoc is previously unknown. Our results provide evidence for the contribution of CNGC-mediated Ca 2+ signaling pathway to this non-host resistance.
In plants, only a few CNGCs such as AtCNGC2 and AtCNGC18 have been proved to function as Ca 2+ channels (Gao et al., 2014;Wang et al., 2017). Whether the tomato CNGC genes encode functional Ca 2+ channels remain to be verified.
In this study, we demonstrated that silencing of SlCNGC1 and SlCNGC14 significantly reduced or almost abolished cytosolic Ca 2+ accumulation (Figure 4). Thus, SlCNGC1 and SlCNGC14 likely function as Ca 2+ channels. Further electrophysiological studies are required to provide more evidences to verify it.
It is recently reported that Arabidopsis cngc2 mutant overaccumulates Ca 2+ in apoplastic compartment and increased HR and resistance (Wang et al., 2017). Similarly, in this study, we found that silencing of SlCNGC1 and SlCNGC14 significantly reduced cytosolic Ca 2+ accumulation and enhanced HR and non-host resistance to Xoc and PTI. Whether silencing of SlCNGC1 and SlCNGC14 results in overaccumulation of Ca 2+ in apoplastic compartment as observed in Atcngc2 mutant (Wang et al., 2017) requires further confirmation.
ROS is the well-known master signal in plant immunity and is indispensable to Xoo-induced HR and non-host resistance . Thus, it is considerable that regulation of ROS accumulation represents one of the essential mechanisms underlying SlCNGC1-and SlCNGC14-dependent regulation of HR and non-host resistance to Xoc and PTI. How SlCNGC1 and SlCNGC14 negatively regulate ROS accumulation remains an intriguing question to be addressed. NADPH oxidase encoded by RBOH genes is the key enzyme to generate ROS during plant-pathogen interactions. Interestingly, Ca 2+ affects the activity of this enzyme which contains EF-hands to bind Ca 2+ . Moreover, AtCPK28, a calcium-dependent protein kinase gene, negatively regulate ROS accumulation during PTI (Monaghan et al., 2014). In this context, it is notable that silencing of SlCNGC1 and SlCNGC14 strongly represses cytosolic Ca 2+ accumulation and expression of a set of denfense-related Ca 2+ signaling genes including SlCaM6, SlCDPK10 and SlCBP60g (Figures 4, 5). Whether and how these changes affect NADPH oxidase thereby modulate ROS accumulation deserves further investigation.
SlCNGC1 and SlCNGC14 belong to group I and group III of tomato CNGC family. They function similarly in negative modulation of tomato HR and non-host resistance to Xoc. Both of them suppress ROS accumulation and callose deposition and alter expression of a same set of defense-related Ca 2+ signaling genes including SlCaM6, SlCDPK10 and SlCBP60g. However, the degree of effect in resistance responses by silencing SlCNGC1 and SlCNGC14 varies (Figures 1-5), indicating the difference of their involvement in this non-host resistance although it can be the consequence of difference of silencing degree. In animal, CNGC often forms homo-or hetero-oligomer to function (Kaupp and Seifert, 2002). Furthermore, some plant CNGC isoforms, such as AtCNGC2 and AtCNGC4, have been reported to be able to homo-or hetero-complex in planta (Chin et al., 2013). In this context, whether SlCNGC1 and SlCNGC14 form oligomer to regulate HR and plant immunity is worth further clarifying. Additionally, cellular localization of plant CNGCs other than long expected plasma membrane have been reported recently (DeFalco et al., 2016b). Whether the cellular localization and accumulation of SlCNGC1 and SlCNGC14 differ awaits further study.
Roles of CNGCs in plant disease resistance seem to be complex. Results from previous studies using Ca 2+ channel blockers indicated that cytosolic Ca 2+ elevation in response to PAMPs such as flg22 is required for PAMP induced defense resposnes including ROS burst (Jeworutzki et al., 2010;Ranf et al., 2011;Segonzac et al., 2011), suggesting a positive role of the possibly involved CNGCs in PTI defenses. However, CNGC genes likely play different roles in various types of resistance. For instance, T-DNA insertion knockout mutants for AtCNGC11 and AtCNGC12 exhibit unaffected flg22 induced PTI responses but show a partial breakdown of resistance against avirulent, but not virulent Hyaloperonospora arabidopsidis and Pseudomonas syringae, indicating that both AtCNGC11 and AtCNGC12 might be not involved in PTI but act as positive regulators of R gene-mediated resistance responses (Yoshioka et al., 2006;Moeder et al., 2011). Differently, null mutants for AtCNGC2 and AtCNGC4 display impaired HR cell death, while maintaining resistance against avirulent pathogens, exhibiting enhanced broad-spectrum resistance against virulent pathogens, elevated levels of SA, and constitutive expression of PR genes (Yu et al., 1998;Clough et al., 2000;Yu et al., 2000;Balagué et al., 2003;Jurkowski et al., 2004;Genger et al., 2008), and thus these AtCNGCs seem to play positive role in HR while have negative role in R gene-mediated resistance and basal resistance. Additionally, AtCNGC2 was reported to positively contribute to Pep-elicited immunity (Qi et al., 2010;Ma et al., 2012Ma et al., , 2013 and LPS-triggered Ca 2+ influx that led to nitric oxide production and consequent HR formation (Ali et al., 2007;Ma et al., 2007), but might be not involved in flg22-triggered immunity (Jeworutzki et al., 2010). In this study, we found the negative role of SlCNGC1 and SlCNGC14 in Xoc-induced HR and non-host resistance, adding further complexity to the function of plant CNGCs. It is likely that different members of CNGC family might have different roles in resistance and at least some CNGCs are multifunctional and differentially regulate various types of resistance against different pathogens. SlCNGC1 and SlCNGC14, which exhibit similar function in Xoc-induced HR and non-host resistance, belong to different groups of CNGCs, with SlCNGC1 to group I as AtCNGC11 and AtCNGC12, while SlCNGC14 to group III, indicating that the function of CNGCs is not group-dependent, as we suggested previously (Saand et al., 2015b). Mechanisms underlying plant CNGC-mediated resistance againt various pathogens await further elucidation.

CONCLUSION
Gene silencing analyses demonstrated that SlCNGC1 and SlCNGC14 negatively regulate non-host resistance related HR cell death and non-host resistance to X. oryzae pv. oryzicola (Xoc) in tomato. These two SlCNGCs repress callose deposition and ROS accumulation to alleviate non-host resistance and likely also PTI. Silencing of SlCNGC1 and SlCNGC14 strongly reduced cytosolic Ca 2+ accumulation, suggesting that SlCNGC1 and SlCNGC14 function as Ca 2+ channels and negatively regulate non-host resistance and PTI through modulating cytosolic Ca 2+ accumulation. SlCNGC14 likely played a stronger regulatory role in the non-host resistance and PTI than SlCNGC1. Our results reveal the contribution of CNGC-mediated Ca 2+ signaling pathway to non-host resistance and PTI.

AUTHOR CONTRIBUTIONS
The project was coordinated by X-ZC. X-RZ conducted the gene silencing analyses. X-RZ and Y-PX carried out the gene expression, designed and analyzed all statistical data. X-ZC conceived of the study, and participated in its design and coordination. X-ZC and X-RZ prepared the manuscript. All authors read and approved the final manuscript.

ACKNOWLEDGMENTS
We are grateful to Prof. Gerald Alan Berkowitz, Department of Plant Science and Landscape Architecture, Agricultural Biotechnology Laboratory, University of Connecticut, United States, for providing seeds of aequorin transgenic tomato lines.