T1N6_22 positively regulates Botrytis cinerea resistance but negatively regulates Pseudomonas syringae pv. tomato DC3000 resistance in Arabidopsis thaliana

ABSTRACT T1N6_22, a short-chain dehydrogenase/reductase family protein, was identified as a positive regulator in Arabidopsis thaliana resistance against Botrytis cinerea and Alternaria brassicae in our preliminary study. In this study, we found that the expression levels of the T1N6_22 gene were induced and up-regulated in A. thaliana ecotype Columbia (Col-0) after B. cinerea and Pseudomonas syringae pv. tomato DC3000 inoculation. Compared with the Col-0 and t1n6_22/T1N6_22 plants, the expression of PAL, PR4, PPO, SOD and CAT genes were down-regulated in the t1n6_22 plants. In Col-0 plants treated with salicylic acid (SA) and the SA analogue benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), the expression levels of T1N6_22 were significantly enhanced, whereas the expression levels of T1N6_22 were reduced by jasmonic acid treatment. Meanwhile, the t1n6_22 mutant exhibited enhanced resistance, whereas the wild-type Col-0 and complemented plants (t1n6_22/T1N6_22) showed susceptibility to Pst DC3000. After inoculation with B. cinerea and Pst DC3000, the expression levels of defence-related genes PR1, PR3, PR5, NPR1 and PDF1.2 in t1n6_22 were significantly different from those in Col-0 and t1n6_22/T1N6_22 plants. Taken together, the T1N6_22 gene played a negative role in Arabidopsis resistance to Pst DC3000. The T1N6_22 gene may be involved in the regulation of salicylic acid and jasmonic-acid–signalling pathways to affect the resistance of Arabidopsis to B. cinerea and Pst DC3000.


Introduction
Grey mould, caused by the fungus Botrytis cinerea, occurs worldwide and causes significant economic losses every year. Isolation and functional analyses of resistance genes related to B. cinerea infection will provide a theoretical basis for elucidation and control of the molecular mechanisms of plant-pathogen interaction. Many reports indicate that plant defence responses against pathogens are mediated by either salicylic acid (SA), or jasmonate/ethylene (JA/ET) signalling pathway [1][2][3]. Tomato plants pretreated with ethylene or methyl jasmonate (MeJA) show decreased susceptibility to B. cinerea. SA accumulation has been reported to be required for local resistance to B. cinerea in Arabidopsis thaliana [4]. Treatment of A. thaliana with exogenous SA could enhance the resistance to subsequent challenge with B. cinerea [5,6]. A. thaliana plants expressing the NahG gene, which inhibits the accumulation of SA, show more susceptibility to B. cinerea than A. thaliana ecotype Columbia (Col-0) [7]. A. thaliana mutants with increased SA levels, showing spontaneous cell death phenotypes, show increased susceptibility to B. cinerea. Concurrent with the hypersensitive response, the SA levels in the inoculated leaves increase [8]. Different defence mechanisms are involved in the response against different pathogens: the SA-dependent response is deployed against the biotrophic pathogens Pseudomonas syringae or Hyaloperonospora parasitica, whereas the JA/ET response is activated by the necrotrophic pathogens B. cinerea or Alternaria brassicicola [9][10][11]. These two molecules play important roles in the regulation of signalling networks in induced defence responses. NPR1 (NON-EXPRESSOR OF PATHOGENESIS-RELATED PROTEINS 1) plays a role downstream in the SA-signalling pathway and also induces the expression of PR1, PR2 and PR5 [12,13]. PR1 (PATHOGENESIS RELATED 1) and NPR1 are commonly used as marker genes that have been found to have a profound impact on the SA-signalling pathway [14,15]. The plant hormones jasmonate and ethylene have been shown to be involved in induced systemic resistance. Plant defence responses have been achieved through activation of genes encoding antimicrobial CONTACT Ji-hong Xing xingjihong2000@126.com; xingjihong@hebau.edu.cn pathogenesis-related proteins or enzymes, which is regulated by jasmonate and ethylene. PDF1.2 (PLANT DEFEN-SIN1.2) and THI2.1 (THIONIN2.1) are marker genes of the JA-signalling pathway [16][17][18].
In the preliminary studies, the T1N6_22 gene, encoding a short chain dehydrogenase/reductase (SDR) family protein, was isolated based on a T-DNA insertion allele. The loss-of-function mutant of T1N6_22 showed enhanced susceptibility to infection by B. cinerea and Alternaria brassicae, suggesting that T1N6_22 was a positive regulator of the basal defence response [19]. In this study, we investigated the function of T1N6_22 in A. thaliana resistance to Pst DC3000 and further analysed the mechanism of T1N6_22 gene regulation in Arabidopsis resistance to B. cinerea and Pst DC3000.

Plant materials and growth conditions
A. thaliana ecotype Columbia (Col-0), T-DNA insertion mutant t1n6_22 and the complemented transgenic plant t1n6_22/T1N6_22 were obtained from the Mycotoxin and Molecular Plant Pathology Laboratory, Hebei Agricultural University. A. thaliana seeds were placed at 4 C for 3 days prior to germination. Plants were grown on soil under fluorescent lights (150 mE¢m ¡2 ¢s ¡1 ) at 22 C with 60% relative humidity and 12h light/12h dark cycle. For axenic growth, seeds were sterilized and sown on a medium solidified with 0.8% agar that contained the salts of Murashige Skoog and 1% (w/v) sucrose. The conditions for axenic growth were 12h light of 60 mE¢m ¡2 ¢s ¡1 .

B. cinerea inoculation
B. cinerea was grown on potato dextrose agar medium and incubated at 20-25 C. Spore inoculums were prepared by harvesting spores in water, filtration through glass wool to remove hyphae and suspension in halfstrength sterile grape juice to a concentration of 4-8 £ 10 6 spores mL ¡1 . Inoculation of A. thaliana plants with B. cinerea was performed on four-week-old plants. Inoculated plants were collected 0, 24 and 48h after inoculation for expression analysis of T1N6_22 and pathogeninducible defence response genes.

Pst DC3000 inoculation
Pst DC3000 was grown in solid King's medium B (KB) medium (containing 25 mg mL ¡1 of rifampicin) at 28 C. A single colony was transferred to 3mL liquid KB medium (containing 25 mg mL ¡1 of rifampicin). The resulting Pst DC3000 liquid culture was agitated at 28 C overnight until mid-log growth phase. The bacteria were harvested by centrifugation at 4000 £ g for 7min, and resuspended in 5mL of 10 mmol L ¡1 MgCl 2 . Four-weekold plants were syringe-infiltrated with Pst DC3000 suspension. To maintain high humidity, the inoculated plants were covered with a transparent plastic film. Inoculated plants were collected 0, 0.5, 1.5, 4 and 8 h after inoculation for expression analysis of T1N6_22 and pathogen-inducible defence response genes.

Colony-forming units assay
A. thaliana leaves inoculated with Pst DC3000 were washed three times with sterile water and were cut into leaf discs of 0.5 cm diameter. Leaf discs were grinded to homogenate with 1mL of sterile water, and then serial dilutions were prepared. One hundred microliters of diluted liquid of leaf homogenate were spread on solid KB plates with 25mg mL ¡1 of rifampicin and were incubated for 2 days at 28 C to score the colony-forming units (CFUs). The experiments were repeated three times and experimental data were analysed by using DPS software.

Hormone treatments
For treatments with signalling hormones, 1 mmol L ¡1 SA (North Tianyi chemical reagent company of Tianjin), 300 mmol L ¡1 benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH; SA analogue, purchased from Trans-Gen Biotech, Beijing, China), 100 mmol L ¡1 JA (purchased from Sigma-Aldrich, United States) and ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC; purchased from Shanghai Dibo Biotechnology Co. Ltd. Shanghai, China) were sprayed on the leaves of fourweek-old plants. The controls were sprayed with distilled H 2 O. Using the method of semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), expression analysis of T1N6_22 was done after treatment with hormones at 0 and 12h. The primers are listed in Table 1. Each treatment was performed at least in triplicate.

RNA extraction, semi-quantitative RT-PCR and quantitative real-time PCR
Total RNA was extracted with TransZol TM Plant reagent (TransGen Biotech, Beijing, China), according to the manufacturer's instructions. The samples were treated with RNase-free DNase to remove the genomic DNA contamination. Two micrograms of total RNA were used as a template for first-strand cDNA synthesis with SuperScript II (Invitrogen, United States) and an oligo (dT) primer.
The semi-quantitative RT-PCR program was the following: 94 C for 4min, 28-33 cycles at 94 C for 30s, 55-60 C for 30s, 72 C for 45 s, and a final elongation step of 5min at 72 C. The PCR products were resolved by electrophoresis in a 2% agarose gel and images were captured by the AlphaImager 3400 system (Alpha Innotech, United States). The A. thaliana housekeeping gene ACTIN was employed for normalization of samples. The ACTIN and pathogen-inducible defence response gene primers are listed in Table 1.
Quantitative real-time PCR was performed using a StepOnePlus System (Applied Biosystems, Germany). Using 10-100ng diluted cDNA as template, amplification was done in 20mL reaction mixture volumes containing 10mL SYBR Premix Taq and 0.8mL 10 mmol L ¡1 primers. The following PCR program was used: 95 C for 30s, followed by 41 three-segment cycles of amplification (94 C for 15s, 56 C for 15s and 72 C for 15s). Primer efficiencies and relative expression levels were calculated using the comparative CT method (User Bulletin 2, ABI Prism 7700 Sequence Detection System). The primers are listed in Table 1. Each treatment had three biological replicates with duplicates for each sample.

Data analysis
The data were analysed by Microsoft Excel and DPS software.

Results and discussion
T1N6_22 gene played a negative role in A. thaliana resistance to Pst DC3000 To study the function of the T1N6_22 gene in A. thaliana resistance to Pst DC3000, the symptoms and bacterial population in the Col-0, t1n6_22 and t1n6_22/T1N6_22 plants inoculated with Pst DC3000 were investigated. It was found that the t1n6_22 plants exhibited enhanced resistance to Pst DC3000, whereas the Col-0 and t1n6_22/T1N6_22 plants showed obvious susceptibility to Pst DC3000 (Figure 1(A)). Consistent with this phenotype, the CFUs of Pst DC3000 in the t1n6_22 plants were significantly lower than those in the Col-0 and t1n6_22/ T1N6_22 plants (Figure 1(B)). The results showed that the t1n6_22 mutant had strong resistance to Pst DC3000, and indicated that the T1N6_22 gene played a negative role in A. thaliana resistance to Pst DC3000.
The expression of T1N6_22 gene was induced by B. cinerea and Pst DC3000 Using the method of quantitative real-time PCR, the expression level of the T1N6_22 gene in Col-0 after B. cinerea and Pst DC3000 inoculation was detected. The results showed that the expression level of the T1N6_22 gene was induced by B. cinerea and Pst DC3000 (Figure 2). In Col-0, B. cinerea infection promoted a ninefold increase (p < 0.05) in the expression of T1N6_22 at  48 h post-inoculation compared with the non-inoculated control. Upon inoculation with Pst DC3000, the expression level of the T1N6_22 gene was 10 times higher (p < 0.05) than that in the non-inoculated control at 8 h postinoculation.

T1N6_22 gene deficiency altered the expression of defence-related genes
To investigate the molecular mechanism of T1N6_22 in plant defence responses, semi-quantitative RT-PCR was used to examine the expression levels of important defence-related genes. Significant (p < 0.05) up-regulation of PAL (L-phenylalanin ammonia-lyase), PR4 (pathogenesis related protein), PPO (polyphenol oxidase), SOD (superoxide dismutase) and CAT (catalase) expression was observed in the t1n6_22 mutant ( Figure 3). In Col-0 and t1n6_22/T1N6_22, the changes in the expression levels of these defence-related genes were basically consistent. These results indicated that the T1N6_22 gene played a negative role in regulating the expression of PAL, PR4, PPO, SOD and CAT.

Expression of the T1N6_22 gene was induced by SA but inhibited by JA
To test the possible relationship of T1N6_22 with the SAdependent and JA/ET-dependent signalling pathways, the effect of exogenous application of a number of signal molecules in Col-0 plants was investigated. In plants treated with SA and the SA analogue BTH, the expression levels of the T1N6_22 gene were significantly (p < 0.05) enhanced. Conversely, the expression level of the T1N6_22 gene was reduced by JA treatment (p < 0.05). The T1N6_22 gene expression level did not change significantly after treatment with the ethylene precursor ACC (Figure 4). These results indicated that the  T1N6_22 gene deficiency alters the expression of key SA-, JA/ET-signalling pathway genes in response to Pst DC3000 To test whether the expression of defence genes in response to Pst DC3000 was affected by T1N6_22 gene mutation, quantitative real-time PCR was used to detect the expression of PR1, PR3, PR5 and NPR1 genes after inoculation with Pst DC3000 (Figure 6). The expression patterns of PR1, PR3, PR5 and NPR1 genes in Col-0 plants were basically consistent. In the early stage of pathogen infection, the expression of PR1, PR3, PR5 and NPR1 genes was induced and the highest levels were observed at 1.5h or 4h post-inoculation; then the expression levels decreased gradually. In t1n6_22 plants, the expression patterns of PR1, PR3, PR5 and NPR1 genes were significantly different (p < 0.05) from those in Col-0 plants: the expression of PR1, PR5 and NPR1 genes was significantly reduced, whereas the expression of the PR3 gene was strongly induced at 4h post-inoculation in t1n6_22 plants. These results suggested that the T1N6_22 gene regulates the expression of PR1, PR3, PR5 and NPR1 genes in the resistance response of A. thaliana against Pst DC3000. It is known that infection of plants by various pathogens triggers a complex battery of defence responses dependent on activation of distinct signalling pathways [20,21]. The SA pathway and the JA/ET pathway are the major molecular mechanisms of plant defence responses. For example, the study of Hyung et al. on the expression of PR1 and PDF1.2 and the content of SA and JA in A. thaliana mutants with enhanced resistance or enhanced susceptibility to B. cinerea implicated RST1 (RESURRECTION 1) as a negative regulator for JA   synthesis or signalling [22]. Two A. thaliana genes, VQ12 and VQ29, which are highly responsive to B. cinerea infection, have been suggested to be partially involved in the JA-signalling pathway and to negatively regulate the plant basal resistance to B. cinerea [23]. GhATAF1, a cotton NAC transcription factor gene, could be highly induced by MeJA, SA and V. dahliae; overexpressing GhA-TAF1 increased cotton plant susceptibility to the fungal pathogens V. dahliae and B. cinerea, coupled with the suppression of JA-mediated signalling and the activation of SA-mediated signalling [24]. Gonorazky et al. [25] reported that tomato SlPLC2 transcript levels were increased after inoculation with B. cinerea. Meanwhile, the transcript levels of the SA-defence pathway marker gene SlPR1a were diminished, whereas the transcripts of SlPI-I and SlPI-II (JA-defence pathway marker genes) were increased in SlPLC2 silenced plants. Rice GF14b is highly expressed during blast infection: GF14b positively regulates panicle blast resistance, and GF14b-mediated disease resistance is associated with the JA-and SAdependent pathway [26]. The tomato SlHUB1 and SlHUB2 genes could be induced by B. cinerea and Pst DC3000 infection and by treatment with SA and ACC; both SlHUB1 and SlHUB2 contribute to the resistance against B. cinerea, most likely through modulating the balance between the SA-and JA/ET-mediated signalling pathways [27].
So far, resistance genes against B. cinerea including BOS1, BOS2, BOS3, BOS4, BIK1 and MYB30 have been cloned from A. thaliana and most of them are regulated by the SA-or the JA/ET-signalling pathway [28][29][30][31][32]. T1N6_22 was identified as a positive regulator of the basal defence response in our preliminary study. In this study, we found that the expression levels of T1N6_22 gene could be induced and up-regulated in wild-type Col-0 plants after B. cinerea and Pst DC3000 inoculation. The expression of key genes of the SA-, JA/ET-signalling pathway in response to B. cinerea and Pst DC3000 infection was altered in t1n6_22 plants. We could speculate that the resistance of A.thaliana to B. cinerea and Pst DC3000 is achieved through expression of defencerelated genes regulated by T1N6_22 in the SA-and JAsignalling pathways. The molecular mechanism of the T1N6_22 gene regulation still needs further study.

Conclusions
The A. thaliana T1N6_22 gene, which encodes an SDR family protein, was shown to be a positive regulator of the basal defence response against B. cinerea infection. In this study, we found that the expression levels of the T1N6_22 gene could be induced and up-regulated in wild-type Col-0 A. thaliana plants after B. cinerea and Pst DC3000 inoculation. In the t1n6_22 plants, the expression of PAL, PR4, PPO, SOD and CAT genes was down-regulated. T1N6_22 was a negative regulator in the response to Pst DC3000 infection. T1N6_22 gene deficiency altered the expression of key genes of the SA-, JA/ET-signalling pathway in response to B. cinerea and Pst DC3000 infection. We could speculate that the T1N6_22 gene may be involved in the regulation of the SA-and JA-signalling pathways to impact the A. thaliana resistance to B. cinerea and Pst DC3000.