Ethylene-mediated resistance to bacterial canker in kiwifruit is suppressed by cool temperature

Abstract

Ambient temperature affects the occurrence and prevalence of plant disease. Most bacterial diseases are damaging at high temperatures. However, kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has been found to be prevalent at relatively cool temperatures, and it is unclear how ambient temperature affects the development of kiwifruit bacterial canker. In this study, basal resistance to Psa was suppressed in kiwifruit at cool growth temperature (16 °C) compared with at normal temperature (24 °C). In addition, RNA sequence analysis and ethylene content assessment indicated that ethylene modulated kiwifruit resistance to Psa at normal growth temperature and that cool temperature inhibited ethylene accumulation and Psa-induced activation of the ethylene signaling pathway in kiwifruit. Virusmediated silencing of the kiwifruit ethylene signaling gene AcEIN2 suppressed kiwifruit resistance to Psa at normal growth temperature. Exogenous application of ethylene inhibitor 1-methylcyclopropene eliminated the difference in kiwifruit resistance to Psa at 16 and 24 °C. Exogenous application of ethylene analogues ethephon induced resistance to Psa in kiwifruit. In conclusion, cool temperatures impair basal resistance to Psa by reducing the activation of ethylene biosynthesis and signaling in kiwifruit. The results provide clues for new strategies to control plant diseases in a context of global environmental change.

Headnote

Received 23 December 2023; Accepted 22 March 2024; Available online 16 August 2024

ABSTRACT

Ambient temperature affects the occurrence and prevalence of plant disease. Most bacterial diseases are damaging at high temperatures. However, kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has been found to be prevalent at relatively cool temperatures, and it is unclear how ambient temperature affects the development of kiwifruit bacterial canker. In this study, basal resistance to Psa was suppressed in kiwifruit at cool growth temperature (16 °C) compared with at normal temperature (24 °C). In addition, RNA sequence analysis and ethylene content assessment indicated that ethylene modulated kiwifruit resistance to Psa at normal growth temperature and that cool temperature inhibited ethylene accumulation and Psa-induced activation of the ethylene signaling pathway in kiwifruit. Virusmediated silencing of the kiwifruit ethylene signaling gene AcEIN2 suppressed kiwifruit resistance to Psa at normal growth temperature. Exogenous application of ethylene inhibitor 1-methylcyclopropene eliminated the difference in kiwifruit resistance to Psa at 16 and 24 °C. Exogenous application of ethylene analogues ethephon induced resistance to Psa in kiwifruit. In conclusion, cool temperatures impair basal resistance to Psa by reducing the activation of ethylene biosynthesis and signaling in kiwifruit. The results provide clues for new strategies to control plant diseases in a context of global environmental change.

Keywords: Kiwifruit; Ethylene signaling; EIN2; Bacterial canker; Plant resistance; Temperature; Pseudomonas syringae pv. actinidiae

1. Introduction

According to disease triangle theory, every plant-pathogen interaction requires appropriate environmental conditions (Wood, 1960). In plant-microbe interactions, environmental conditions affect both parties simultaneously (Kuruppu et al., 2024). The optimal in vitro growth temperature for many Pseudomonas syringae is 28 °C. However, high temperatures usually negatively affect the production of their virulence factors (e.g., phytotoxins, extracellular poysaccharide, or type-II secreted effector secretion), though almost all of the studies have been conducted in vitro (van Dijk et al., 1999; Smirnova et al., 2001; Weingart et al, 2004; Puttilli et al, 2022). In Arabidopsis, high temperature (30 °C) promotes the secretion of type III effectors from P. syringae pv. tomato DC3000 to the host (Huot et al., 2017). The mechanisms by which temperature affects Pseudomonas virulence in plants remains unclear, and different Pseudomonas virulence factors may respond to temperature in different ways (Li et al., 2019). Temperature is an important environmental factor that regulates plant growth and development as well as interactions with other organisms, and changes in ambient temperature often affect plant defense responses (Hua, 2014). Temperature changes can regulate NLR (the nucleotide-binding oligomerization receptors) genesmediated resistance by altering NLR proteins expression level and stability (Yang et al., 2010; Zhu et al, 2010; Cheng et al., 2013). In addition, pattern-triggered immunity (PTI) response is also regulated by changes in ambient temperature. In Arabidopsis leaves or protoplasts, the optimal temperature for flg22 to activate the PTI marker genes WRKY29 and FRK1 is approximately 28 °C, and the induction is significantly attenuated at temperatures below 16 °C (Cheng et al., 2013). Furthermore, flg22 activates MAPK (mitogen-activated protein kinase, the key components of basal resistance) more rapidly at 28 or 23 °C than at 16 °C (Cheng et al., 2013; Bigeard et al., 2015). The molecular mechanisms by which temperature regulates basal resistance and the effects of temperature changes on basal resistance in other plant species especially ligneous plants require further explored.

Temperature may regulate plant resistance by affecting hormones, including salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and ethylene (ET) (Denancé et al., 2013). SA plays an important role in temperature-regulated plant immunity (Kim et al., 2013; Mine et al., 2017). The effector-induced increase in SA is suppressed at high temperatures. It has been shown that 30 °C inhibits SA biosynthesis mediated by the key synthase ICS1 (isobranching synthase 1) (Huot et al., 2017). At the same time, elevated temperatures affect SA-dependent gene expression by activating SA-negative regulators. For example, the expression level of МУС2 (a master regulator of JA signaling and a negative regulator of SA signaling) was higher at 30 °C than at 22 °C (Huot et al., 2017). On the other hand, low temperatures enhanced the SA pathway to promote immunity, possibly by inhibiting SA biosynthesis via ethylene (Li et al., 2019). The molecular mechanisms by which temperature regulates plant resistance differ markedly in different plant-pathogen interaction systems. In rice, the JA-dependent transcription factors OsMYC2 and OsMYB22 can synergistically regulate the expression of OsCEBiP (basal resistance gene), thereby regulating basal resistance (Qiu et al., 2022). At 22 °C, Magnaporthe oryzae was not able to effectively induce JA biosynthesis and signaling, which resulted in increased susceptibility of rice to M. oryzae (Qiu et al., 2022). The mechanism of how various phytohormones coordinate to regulate plant resistance at different temperatures is needs further investigation.

Kiwifruit bacterial canker (KBC), caused by P. syringae pv. actinidiae poses a serious threat to kiwifruit production. The pathogen is distinguished by its wide range, rapid spread, and strong virulence. It has the potential to cause numerous tree fatalities within a brief timeframe, rendering prevention and control efforts challenging (Gao et al., 2016; Kuang et al., 2024). The disease was first detected in Japan in 1984, and was subsequently found in France, New Zealand, Korea, Iran, Italy, Portugal, Chile, and China, causing severe economic losses to the kiwifruit industry (Serizawa et al., 1989; Koh et al., 1994; Scortichini, 1994; Mine et al, 2017; Su et al, 2025). The timing of KBC outbreaks and the extent of damage were closely linked to temperature and rainfall (Beresford et al., 2017). Cool temperatures and high humidity favor the invasion and rapid multiplication of the pathogen. In Japan, kiwifruit branches inoculated with Psa did not develop spots at average temperatures > 20 °C (Serizawa and Ichikawa, 1993). Using GFP to label Psa, Psa spread more rapidly in kiwifruit branches and leaf veins at cool temperatures (16 °C) compared to 24 °C (Gao et al, 2016). Notably, the preference of Psa for cool temperatures differs sharply from the preference of common bacterial diseases for higher temperatures. Several studies have shown that kiwifruit defense against Psa is mainly associated with SA-mediated pathways, while JA, ET, and ABA are also involved in the regulation of defense against Psa (Petriccione et al., 2013; Reglinski et al., 2013; Cellini et al., 2014; Nunes da Silva et al., 2021). However, the current study was mainly conducted at temperatures below 20 °C. It is unclear whether the increased resistance of kiwifruit to Psa at normal growth temperature (24 °C) is related to temperature-regulated hormone levels and signaling.

In this study, we investigated the impact of ambient temperature on the development of KBC. Subsequently, the mechanism by which cool temperature inhibits kiwifruit basal resistance was elucidated through transcriptomic and genetic analyses. This provides a theoretical foundation for the development of efficacious strategies for the management of KBC.

2. Materials and methods

2.1. Plant materials and pathogen inoculation

Two-year-old susceptible varieties of kiwifruit trees, Actinidia chinensis var. chinensis 'HongYang', were cultivated in a greenhouse at Northwest Agriculture and Forestry University, Shaanxi Province, China. Leaves and branches were collected for pathogenicity determination. The strongly pathogenic Psa strain M228 was isolated from infected leaves of 'HongYang' in Shaanxi Province, China (Zhao et al, 2019). M228 was cultured with Luria-Bertani (LB) plates at 28 °C.

Before inoculation with Psa, all the kiwifruit plants were acclimatized. Branches and leaves were kept for 3 days at 4, 10, 16, 24, 30 and 37 °C, respectively. Histocultured kiwifruit seedlings were transferred respectively to incubator for 1 week at 16 and 24 °C. For branch inoculation, healthy kiwifruit branches without Psa were selected and cut into 50 cm lengths. Surface disinfection was performed with 0.6% sodium hypochlorite for 10 min, then washed three times with sterile water, dried naturally, and carefully cut with a sterilized scalpel. Then 10 pL of 2 x 10° СРО. mL · bacterial suspension was inoculated into each wound area. The inoculated kiwifruit branches were placed in an incubator at the corresponding temperature, respectively. Disease spot length was measured 15 days after inoculation. At least three independent experiments were performed.

We inoculated Psa into kiwifruit leaves using the vacuum infiltration method (Zhao et al., 2019). Surface-sterilized healthy leaves were punched out of the leaf discs with a 10-mm-diameter punch and immersed in 30 mL of bacterial inoculum in a 50-mL tube. Vacuum penetration was carried out using a vacuum pump and a glass cover until the leaf disk sank to the bottom of the tube. Place 10 evenly infiltrated leaf discs into 0.1% water agar petri dishes and incubate in a dark incubator at 16 °C or 24 °C. The phenotypes were observed and photographed at the 5 days post inoculation (dpi). The bacterial growth assay method was referred to a previously described procedure (Yuan and Xin, 2021). For bacterial growth counting, a pDSK-GFPuv plasmid with a kanamycin resistance screening marker was expressed in Psa bacteria (Gao et al., 2016).

2.2. Virus-induced gene silencing of kiwifruit leaf

The TRV-based VIGS assay was used to transiently silence AcEIN2 in this study. To avoid possible silencing by other homologs, a specific fragment of AcEIN2 was amplified from kiwifruit cDNA library using gene-specific primers (Table S1) and cloned into the VIGS vector pTRV2. Agrobacterium rhizogenes strains carrying the pTRV1 vector were mixed in a 1 : 1 ratio with constructs containing pTRV2-AcEIN2, pTRV2-GFP, and pTRV2AcPDS, respectively. All kiwifruit histocultures were removed after 90 d of rooting culture, rinsed with sterile water, and then placed in the prepared Agrobacterium infiltration mixture so that all leaves were completely submerged. The seedlings were then vacuum infiltrated as described above, rinsed with sterile water, dried with sterilized filter paper, and cultured in fresh rooting medium. After 15 days, samples were collected and the efficiency of gene silencing was determined by qRT-PCR.

2.3. RNA-seq and qRT-PCR analysis

Total RNA was isolated from three independent biological replicates using Quick RNA Isolation Kit (HuaYueYang, Beijing, China) according to the manufacturer's schedule. For whole transcriptome analysis, total RNA was processed using NovaSeq 6000 (Illumina) from Novogene (Tianjin, China) to obtain clean reads. Clean reads were mapped to the reference genome of A. chinensis 'HongYang' v3 using HISAT2 software (Wu et al., 2019; Zhang et al., 2021). In addition, pathway analysis was performed by downloading the A. chinensis database in Mapman (https:// mapman.gabipd.org/).

gRT-PCR was used to determine relative gene expression levels, and Table S1 lists all gene-specific primers used for qRTPCR. Briefly 1 pg of total RNA was synthesized as a template for first-strand cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Massachusetts, USA), and then quantitative reverse transcription was performed using ChamQTM SYBR® qPCR Master Mix (Vazyme, 0311, Nanjing, China) for qPCR. The internal control gene actin was used for normalization. The relative expression levels of three technical replicates were evaluated by the 24" method.

2.4. Measurement of H,O, and callose

For H,0, detection, three replicate kiwifruit leaf discs from each set of samples were placed in DAB staining solution and incubated under light for 8 h. After the DAB solution was absorbed, the discs were decolorized in a decolorizing solution (anhydrous ethanol: acetic acid, 1:1, v/v) until the samples became transparent. To observe callose deposition, postinoculated kiwifruit leaves were decolorized in the decolorizing solution described above and then soaked in chloral hydrate overnight. Transparent leaves were stained with 0.05% aniline blue in 0.067 mol - L' K,HPO, (pH 9.6). Representative photographs were taken using a microscope (Olympus, Tokyo Japan). The content of H,0, in kiwifruit leaves was detected using spectrophotometry, as H,O, and titanium sulfate produce a yellow titanium peroxide complex with characteristic absorption at 415 nm. The content of callose in kiwifruit leaves was detected using the fluorescence method, as callose reacts with aniline blue dye to produce fluorescent substances. All reagents were purchased from Suzhou Comin Biotechnology, and the experimental procedures were conducted according to the manufacturer's instructions (Han et al., 2023).

2.5. Western blot assays

The kiwifruit leaves were ground into a fine powder in liquid nitrogen to extract the total proteins using the extraction buffer [25 mmol - 1,7! Tris-HCl (pH 7.4), 150 mmol - Lİ NaCl, 1 mmol - L7· EDTA, 1% Nonidet P-40, 5% glycerol] with 1 mmol - 171 phenylmethylsulfonyl fluoride (PMSF), and Protease inhibitor mixture (Solarbio). The total proteins were then resolved in 10% SDS-PAGE gels and immunoblotted with anti-Phospho-p44/42 MAPK (Cell Signaling Technology) and anti-Actin for plant (Abmart).

2.6. Plant phytohormone quantification

The contents of 1-aminocyclopropane-1-carboxylic acid (ACC, a direct biosynthetic precursor of ethylene) was determined as previously described and modified (Qiu et al., 2022). SA and JA were extracted as described previously (Pan et al., 2010). Briefly 0.1 g of fresh leaf was rapidly frozen and ground in liquid nitrogen. The resulting powder was transferred to a 5 mL centrifuge tube with 2 mL of extraction solvent (2-propanol/H,0/concentrated HC], 2 : 1: 0.002, v/v/v). The mixture was shaken at 100 г - min · for 30 min at 4 °C. Next, 2 mL dichloromethane was added and mixed again for shaking additional 30 min at 4 °C. The sample was then centrifuged at 13 000xg for 5 min to form two phases, and 2 mL lower phase liquid was transferred to a vial and concentrated (incompletely dried) using a nitrogen evaporator. Then the last mixture was redissolved in 0.2 mL of methanol and 50 uL sample was taken to detect plant hormones by Nexera UHPLC LC-30A (Shimadzu, Japan).

2.7. Exogenous ethephon and 1-MCP treatment

Surface-sterilized healthy kiwifruit leaf discs were obtained with a 10-mm diameter punch and placed on water agar medium. Then 2 ul - L7· of 1-MCP or 500 mg - L · of ethephon was sprayed uniformly on the leaves, and petri dishes were sealed. The treated leaf discs were placed in a 16 °C or 24 °C incubator for dark culture, and after 2 d, they were inoculated with Psa as described above.

3. Results

3.1. Ambient temperature strongly affects KBC development

To determine the effect of ambient temperature on disease development, we conducted infestation tests on kiwifruit branches and leaves under different temperature conditions. Leaves were inoculated with the highly virulent Psa strain M228 via vacuum infiltration and incubated at temperatures ranging from 4 °C to 37 °C. The bacterial propagation assay indicated that Psa colonization was the most favorable at 16 °C, while colonization was inhibited at 4 and 37 °C. Although Psa can reproduce at 10 and 30 °C, it cannot cause visible disease spots (Fig. 1, A). Kiwifruit branches inoculated with M228 were incubated at 16 and 24 °C, respectively. Measurements of spot length 15 days after inoculation showed that Psa caused significantly more spots at 16 °C than at 24 °C (Fig. S1, A, B). Moreover, similar results were observed in the resistant varieties A. chinensis var. deliciosa 'Hayward' (HWD), A. chinensis var. deliciosa 'Xuxiang' (XX), A. chinensis var. deliciosa 'Cuixiang' (CX), and the new yellow-fleshed variety A. chinensis var. chinensis 'Nongda Jinmi' (NDJM) (Fig. 1, В and ©). All these results indicate that ambient temperature strongly fluences the of KBC.

3.2. Cool temperature does not reduce Psa replication

We then examined whether the increased pathogen propagation in plants at 16 °C compared to 24 °C was due to increased pathogen replication at lower temperatures. First, we analyzed the growth of Psa M228 at 16 and 24 °C in LB liquid medium. Before 48 h, the density of Psa M228 at 24 °C was higher than at 16 °C, but at 48 h they were similar. After that, the density of bacteria at 16 °C was slightly higher than at 24 °C, with little difference at 120 h (Fig. 2, A). Subsequently, we measured the number of bacteria in kiwifruit leaves at different time points. At 1d, there was no significant difference in the number of Psa M228 populations inoculated at 16 and 24 °C. After 1 day, Psa M228 populations were larger at 16 °C than at 24 °C. By 5 days after inoculation, pathogen propagation was more than 10 times greater at 16 °C than at 24 °C (Fig. 2, B). Therefore, greater pathogen multiplication at 16 °C than at 24 °C cannot be attributed to differences in the intrinsic growth rate of the pathogen at different temperatures.

3.3. Basal resistance induced by Psa is impaired in cool temperature

Although temperature regulates effector-triggered immunity (ETI) responses induced by bacterial effectors, plants preferentially activate ETI signaling at relatively lower temperatures (10-23 °C) (Cheng et al., 2013). However, our tests in several kiwifruit cultivars suggest that cool temperature inhibition of resistance in kiwifruit may be universal. Furthermore, Psa M2284hrcC, which is unable to deploy a type Ш effector (Zhao et al., 2019), also reproduces more at 16 °C than at 24 °C within kiwifruit leaves, consistent with Psa M228 (Fig. 52). This suggests that the increase in kiwifruit susceptibility at cool temperatures may not be caused by impairment of R gene-mediated resistance. Therefore, we hypothesized that the basal resistance of kiwifruit may be impaired at 16 °C. We determined markers of basal resistance responses in kiwifruit at different temperatures to test this hypothesis. Kiwifruit leaves inoculated with Psa were incubated at 16 and 24 °C for 24 h. The aniline blue staining and callose content assay showed that the callose content in kiwifruit leaves was lower at 16 °C (Fig. 3, A and B), and the amount of Psainduced H,O, in kiwifruit leaves at 16 °C was also significantly lower than at 24 °C (Fig. 3, C and D). Similarly, MAPK activity in Psa-inoculated kiwifruit leaves was lower at 16 °C than at 28 °C (Fig. 3, E). The defense-related genes AcPSL5, AcWRKY22, and АсРТ15 were also not significantly induced by Psa inoculation at 16 °C (Jing and Liu, 2018; Liu et al., 2022) (Fig. 3, F). Collectively, these results suggest that basal resistance to Psa in kiwifruit is more impaired at 16 °C than at 24 °C.

3.4. Cool temperature inhibits ethylene biosynthesis and the expression of signaling genes in kiwifruit

To further elucidate the mechanisms underlying this temperature-dependent basal resistance in kiwifruit, RNA-seq was performed to determine the effect of temperature on global transcriptional reprogramming in kiwifruit during Psa infection. After vacuum infiltration inoculation with Psa, kiwifruit leaves were placed at 16 and 24 °C, respectively, and samples were collected at 24 h to avoid the effect of different bacterial population densities in the plants, and subsequently analyzed by RNA-seq (NCBI, PRINA1077644). The reads obtained were used to identify differentially expressed genes (DEGs) using a four-fold change in expression as a cut-off criterion (log, value > 2, P value < 0.01). A total of 2228 DEGs responded to temperature, of which 1447 were up-regulated and 758 were down-regulated (Fig. 53, A). KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of kiwifruit at 16 °C vs. at 24 °C was performed for all the DEGs to identify the major pathways involved in the response to temperature. DEGs were primarily involved in "Plant hormone signal transduction", "Biosynthesis of amino acids", and "Plant-pathogen interaction" (Fig. 53, В).

To further understand the pathways involved, we analyzed the DEGs using Mapman software. Psa specifically induced ET pathway-related genes at 24 °C, suggesting that ethylene biosynthesis and signaling pathways may be involved in the temperature-dependent bacterial canker resistance of kiwifruit (Fig. S3, C). In general, ethylene plays an important role in plant disease resistance (Zhao et al., 2020). Since ethylene signaling pathway genes were enriched in DEGs in the transcriptome analysis, qRT-PCR analysis was performed to verify the expres sion levels of several ethylene biosyntheses and signaling path ways genes. Consistent with the results of RNA-seq analysis, the expression of AcACO7, AcACS2, AcEIN2, AcEIN3 and AcEIL1 was preferentially activated at 24 C than 16 C(Fig. 4, A). The differ ential expression levels of these genes suggest that ethylene accumulationinkiwifruitmayalsobedifferentat16and24 C.To assess ethylenelevels, wemeasuredthelevelsofACC.ACClevels were higher in kiwifruit plants inoculated with Psa at 24 C, and Psa-induced ACC synthesis was inhibited at 16 C (Fig. 4, B). Similarly, kiwifruit branches inoculated with Psa were placed at 16 and 24 C, and samples taken after 24 h for ACC content showedthatACCcontentwassignificantlyhigherat24 Cthanat 16 C. There was no significant difference in ethylene content in branches not inoculated with Psa (Fig. S4). Psa-induced SA and JA synthesis was not inhibited at 16 C(Fig. 4, B). All these results suggest that the ethylene pathway may be involved in the temperature-dependent regulation of bacterial canker resistance in kiwifruit.

3.5. Inhibition of ethylene signaling enhances kiwifruit's sensitivity to Psa

In Arabidopsis, the ethylene receptor located in the endoplasmic reticulum (ER) activates a Ser/Thr kinase called Constitutive Triple Response 1 (CTR1), which negatively regulates the ET response by phosphorylating Ethylene Insensitive 2 (EIN2), an ER-bound protein. ET binding to receptors inactivates CTR1, which dephosphorylates EIN2, allowing it to translocate to the nucleus (Kieber et al., 1993; Gao et al., 2003; Huang et al, 2003; Ju et al., 2012; Lacey and Binder, 2014). In the nucleus, EIN2 activates the transcription factor EIN3 and the ethylene insensitive-like protein 1 (EIL1), which then binds to the promoters of various target genes to regulate their expression (Solano et al., 1998; Qiao et al., 2009; An et al., 2010; Ju et al., 2012). To investigate the role of ethylene signaling in increasing kiwifruit susceptibility at cool temperatures, we silenced AcEIN2 using the TRV2-mediated VIGS technique in 'HongYang' kiwifruit and performed Psa infection assays. Plants were more susceptible to Psa after AcEIN2 silencing at 24 °C. In AcEIN?-silenced plants, the differences in disease severity at different temperatures were less significant than in the corresponding GFP-silenced plants (Fig. 5, A and B). AcEIN2 was effectively silenced as determined by qRT-PCR analysis (Fig. 5, C). In addition, the differences in Psa-induced expression of AcPSL5, ACWRKY22 and АсРТ15 were less significant in AcEIN2silenced plants than in GFP-silenced plants at different temperatures (Fig. 5, D). H,O, levels induced by Psa were also significantly reduced in AcEIN2-silenced plants, and the differences were significantly smaller than those in wild-type plants at different temperatures (Fig. S5, A, B). These results suggest that cool temperature impairs the induction of ethylene signaling by Psa, resulting in reduced basal resistance in kiwifruit.

To further investigate the effect of ethylene on temperature-regulated bacterial canker resistance, kiwifruit leaves were treated in an infection assay with 2 pL - L7· 1-MCP (an inhibitor of ethylene perception), which binds tightly to the ethylene receptor and blocks ethylene perception, and 500 mg - LA ethephon, which releases ethylene and induces ethylene production. There were no significant differences in Psainduced lesion area and bacterial colonization after 1-MCP treatment compared with the control at 16 °C. However, bacterial colonization was significantly higher after 1-MCP treatment at 24 °C compared to the control, indicating that 1-MCP treatment at 24 °C could suppress the resistance of kiwifruit. At 16 and 24 °C, both lesion area and bacterial colonization were reduced in ethephon-treated kiwifruit leaves compared with the control, suggesting that ethephon treatment could induce resistance. In addition, the difference in lesion area and bacterial colonization caused by Psa was significantly lower after 1-MCP treatment compared to H,O treatment at both temperatures (Fig. 6, A and B). These results suggest that an important reason for the increased incidence of bacterial canker in kiwifruit is the impaired ethylene accumulation at 16 °C.

4. Discussion

In crop production, abiotic stresses and pathogens are two major factors that negatively affect yields and can lead to significant losses. Elevated ambient temperatures may be a key aspect of climate change in the coming decades, which will likely have a major impact on global crop production (Bebber et al., 2013; Bebber, 2015; Zhou et al., 2023). Elevated temperatures have a tendency to disrupt the immune response of the plant and encourage the infestation of pathogenic bacteria. High field temperatures favor the development of bacterial blight, and disease severity is particularly high during the hot season (Webb et al., 2009). In contrast, the incidence is lower in the cool season. Cyanosis caused by Ralstonia solanacearum, for example, is more severe in Solanaceae under high temperature and humidity conditions (Yang et al., 2023). It has also been shown that relatively high temperatures in the range of 16-30 °C inhibit salicylic acid-mediated immune responses and promote DC3000 infection in A. thaliana (Huot et al., 2017; Li et al., 2019). In contrast, in this study, Psa, a pathogenic variant of P. syringae, infects kiwifruit more readily at cool temperatures. But in vitro culture, Psa was not found to be more adapted to cool temperatures (Fig. 2, A). In addition, basal resistance in kiwifruit mediated by the ethylene pathway was suppressed at cool temperatures, which may be the reason why cool temperatures are more favorable for KBC development.

In response to pathogen attack, the plant immune system has developed two levels of response (Jones and Dangl, 2006). The first level of defence senses conserved pathogen-associated lecular (PAMP), leading to PTI. The second level consists of intracellular NLRs that specifically detect effector virulence activity and activate a strong innate immune response known as ETI (Wang et al., 2019). Temperature has an important effect on both tiers of responses in plant immunity. In Arabidopsis, flg22induced basal resistance responses occurred faster and were stronger at 28 or 23 °C than at 16 °C (Cheng et al., 2013). Similarly, in this study, H,O, and callose induced by Psa, as well as the expression of PTI-related genes (AcPSL5, ACWRKY22, AcPTI5), were suppressed in kiwifruit at 16 °C compared with at 24 °C, suggesting that the basal immune response was suppressed in kiwifruit at 16 °C (Fig. 2, A-F). In many pathosystems, high temperatures typically inhibit plant ETI. For example, the tobacco N protein used for resistance to tobacco mosaic virus (TMV) (Whitham et al., 1996), and several tomato Cf proteins used for resistance to the leaf mold pathogen Cladosporium fulvum do not produce effective ETIs at temperatures higher than 30 °C (de Jong etal., 2002). In addition, the disease resistance and HR induced by Pst carrying avrRpt2 or avrRpm1 were more significantly reduced at 28Cthanat22C(Wangetal.,2009).ETIsignalingmediatedby the Arabidopsis NLR proteins RPM1 and RPS2 is also temperature regulated and peaks at 16 C(Cheng et al., 2013). However, in contrast to most R proteins, Xa7, a rice disease resistance protein targeting Xanthomonas oryzae, was more effective at higher tem peratures (Webb et al., 2009). This study indicates that among different kiwifruit cultivars, lower temperatures are more conducive to the invasion of Psa. There were also significant differences in the colonization of the Psa mutant DhrcC(unable to secrete type III effectors) in kiwifruit leaves under two different temperatures (Fig. S2, B). Therefore, we speculate that the decrease in resistance of kiwifruit to Psa at cool temperatures is not related to R gene mediated resistance.

The role of ET in plant immune response is complicated, and whether ET positively or negatively regulates plant defense response may depend on specific environmental conditions and plantepathogen interaction systems (Hoffman et al., 1999; Geraats et al., 2003; van Loon et al., 2006; Washington et al., 2016; Yang et al., 2018). For example, the accumulation of FLS2 (Flagellin Sensitive 2) (receptor for bacterial flagellin protein or its active epitope Flg22) is reduced in ET-insensitive etr1 and ein2 mutants (Boutrot et al., 2010; Mersmann et al., 2010; Tintor et al., 2013). Arabidopsis ein2 mutants showed increased sensitivity to Erwinia carotovora (Chen et al., 2009). Similarly, ET can increase disease resistance in poplar by polar PR genes and H2O2 produc tion (Liu et al., 2022). Previous studies analyzing kiwifruit defense mechanisms against Psa by RNA-seq did not find significant dif ferences in ethylene pathway-related genes (Song et al., 2019; Li et al., 2020; Nunes da Silva et al., 2021). However, these studies were conducted at temperatures below 20 C. Our study found that the biosynthesis and signal transduction of ET-related genes such as ACS1, EIN2, and EIL1 induced by Psa in kiwifruit were significantly inhibited at 16 C(Fig.4,AeF).Atthesametime,ACC synthesis in kiwifruit was significantly reduced at 16 C, but Psa can still induced SA and JA synthesis (Fig. 4,GeI). In addition, we silenced the key factor of ET signaling, EIN2, in kiwifruit and found that the basal resistance response of EIN2-silenced kiwi fruit was significantly reduced at 24 C and sensitivity to Psa was increased, but there is no significant change at 16 C(Fig. 5). Exogenous application of the ET inhibitor 1-MCP increased the sensitivity of kiwifruit to Psa at 24 C, while the use of ET analogs ethephon increased the resistance of kiwifruit to Psa at 16 C (Fig. 6). The impact of temperatures on the immune response of plants remains poorly understood. Arabidopsis resistance to DC3000 could be increased as the temperature decreased within the range of 16e30 C. Especially, the increased resistance was primarily due to SA mediated immune response at low temper atures (Huot et al., 2017; Li et al., 2019). Differently, a warm temperature (22 C) suppresses rice basal resistance to M. oryzae more than at 28 C, which is related to JA biosynthesis and signaling (Qiu et al., 2022). Interestingly, in kiwifruit, a perennial horticultural plant, temperature regulates resistance to Psa by affecting the ethylene pathway. This indicates that temperature affects plant resistance through specific signal pathways in different plantepathogen interactions.

In conclusion, ethylene mediates resistance to Psa in kiwifruit at normal growth temperature (24 C), and ethylene biosynthesis and signaling are inhibited at cool temperatures (16 C), resulting in increased susceptibility to Psa in kiwifruit. In addition, the moderate use of ethephon under cool temperature may be effective to control Psa under cool temperature. However, the effect of temperature on Psa pathogenicity was not analyzed in this study, and it cannot be excluded that cool temperature induced the expression of Psa virulence factors, such as effectors or toxins. Further studies are needed to investigate the expres sion of Psa virulence factors under the dual effects of host plant environment and temperature changes.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by grants from the National Key Research and Development Program of China (Grant No. 2022YFD1400200) and the Special Support Plan for High-Level Talent of Shaanxi Province.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.1016/j.hpj.2024. 03.008.

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Footnote

1 These authors contributed equally to this work.

* Corresponding author.

E-mail address: huanglilienwafu.edu.cn

Peer review under responsibility of Chinese Society of Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS)