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. 2024 Jun 3;119(3):1353–1368. doi: 10.1111/tpj.16855

A multifaceted crosstalk between brassinosteroid and gibberellin regulates the resistance of cucumber to Phytophthora melonis

Yunyan Kang 1,#, Zhongli Jiang 1,#, Chen Meng 1, Xianpeng Ning 1, Gengzheng Pan 1, Xian Yang 1,✉, Min Zhong 1,✉
PMCID: PMC13087484  PMID: 38829920

SUMMARY

Cucumber plants are highly susceptible to the hemibiotroph oomycete Phytophthora melonis. However, the mechanism of resistance to cucumber blight remains poorly understood. Here, we demonstrated that cucumber plants with impairment in the biosynthesis of brassinosteroids (BRs) or gibberellins (GAs) were more susceptible to P. melonis. By contrast, increasing levels of endogenous BRs or exogenously application of 24‐epibrassinolide enhanced the resistance of cucumber plants against P. melonis. Furthermore, we found that both knockout and overexpression of the BR biosynthesis gene CYP85A1 reduced the endogenous GA3 content compared with that of wild‐type plants under the condition of inoculation with P. melonis, and the enhancement of disease resistance conferred by BR was inhibited in plants with silencing of the GA biosynthetic gene GA20ox1 or KAO. Together, these findings suggest that GA homeostasis is an essential factor mediating BRs‐induced disease resistance. Moreover, BZR6, a key regulator of BR signaling, was found to physically interact with GA20ox1, thereby suppressing its transcription. Silencing of BZR6 promoted endogenous GA biosynthesis and compromised GA‐mediated resistance. These findings reveal multifaceted crosstalk between BR and GA in response to pathogen infection, which can provide a new approach for genetically controlling P. melonis damage in cucumber production.

Keywords: brassinosteroids, gibberellin, cucumber, hormone interactions, Phytophthora melonis

Significance Statement

This study demonstrates that GA homeostasis is necessary for the BRs‐mediated resistance of cucumber to Phytophthora melonis. BZR6, a key regulator of BR signaling, directly suppresses the transcription of GA20ox1 and consequently reduces GA1 and GA3 contents. In conclusion, our results reveal the multifaceted regulation mechanism of BR/GA cooperation in growth–defense trade‐offs.

INTRODUCTION

Phytophthora blight has been pronounced as a destructive disease of Solanaceae and Cucurbitaceae vegetable crops worldwide. Infection can occur at any plant stage, causing root, crown, and fruit rot, along with leaf blight (Reis et al., 2018; Wu et al., 2016). Phytophthora root and crown rot cause lesions on the roots and lower stems, leading to stem girdling, plant wilting, and ultimately plant death (Quesada‐Ocampo et al., 2023). Great efforts have been made to screen resistant lines or resistance‐related genes in Cucurbitaceae (Ayala‐Donas et al., 2022; Donahoo et al., 2013; Hashemi et al., 2020; Kousik et al., 2021; LaPlant et al., 2020; Wu et al., 2014), and to develop molecular markers based on quantitative trait loci associated with resistance to root and crown rot (Ramos et al., 2020; Vogel et al., 2021). However, the markers identified to date have not been successfully bred into cultivars with marketable standards, and the poor horticultural performance of resistant lines has limited their application in production. Therefore, overcoming the damage caused by root and crown rot disease remains the primary focus of breeding efforts in Cucurbitaceae production.

Brassinosteroids (BRs) are a group of polyhydroxylated plant steroid hormones that play vital roles in plant growth and development (Li et al., 2020; Nolan et al., 2020). Brassinolide (BL) and its immediate precursor castasterone (CS) are two of the most biologically active BRs identified in higher plants to date. The CYP85A in the cytochrome P450 family catalyzes the C‐6 oxidation of 6‐deoxocastasterone to CS and the subsequent conversion of CS to BL, which plays a crucial role in the regulation of biologically active BR levels (Kwon & Choe, 2005). Binding of active BR to the receptor brassinosteroid‐insensitive 1 (BRI1) triggers a series of transduction events, eventually leading to the dephosphorylation and activation of Brassinozole Resistant 1/bri1 EMS‐Suppressor 1 (BZR1/BES1). Finally, the dephosphorylated BZR1/BES1 regulates the expression of BR target genes to elicit specific physiological processes (Clouse, 2011).

Beyond their primary functions in the plant growth and development, BRs are also involved in growth‐defense trade‐offs. The BR signaling pathway exhibits cross‐talk with various immune‐related pathways triggered by diverse microbe‐ and pathogen‐associated molecular patterns at the receptor, cytoplasmic, or transcriptional level (Antolín‐Llovera et al., 2012; Bar et al., 2010; Bjornson et al., 2016; Chen & Shimamoto, 2011; Feng et al., 2021; Kalachova et al., 2022; Lin et al., 2014; Ranf et al., 2012; Saini et al., 2022; Turnbull et al., 2019; Yang et al., 2007; Yu et al., 2018; Zhang & Zhou, 2010). In Nicotiana benthamiana–Phytophthora sojae interactions, the virulence effector aldose 1‐epimerase (AEP1) activates plant immunity through mediating P. sojae extracellular sugar uptake, which depends on the BR receptor brassinosteroid insensitive 1‐associated receptor kinase 1 (BAK1) (Xu et al., 2021). AVR2, an RXLR effector secreted by the potato blight pathogen Phytophthora infestans, suppresses cell death triggered by the elicitin Infestin 1 through activating the expression of the BR‐responsive marker gene StCHL1 (Turnbull et al., 2017). The cytoplasmic fraction of BZR1 was also confirmed to positively regulate effector‐triggered immunity mediated by the resistance protein RPS4 in the TIR‐NB‐LRR protein class; however, this effect was weakened with the nuclear translocation of BZR1 (Qi et al., 2021). In addition, the tomato BR‐insensitive mutant curl3 exhibited reduced disease resistance against P. infestans (Dallagnol et al., 2023). Overexpressing BES1 could enhance the resistance of apple to Valsa mali attack (Liu et al., 2021). Our previous study also confirmed the suppressive effect of 24‐epibrassinolide (EBR) on Phytophthora melonis infection through regulating the development of the hypocotyl and vascular bundle in cucumber plants (Ren et al., 2020).

BRs and gibberellins (GAs) are two important phytohormones that have similar effects on several processes of plant growth and development. BR‐ and GA‐deficient mutants show similar phenotypes to varied degrees, including decreased seed germination, de‐etiolation in the dark, dwarfism, and delayed flowering. The coordination and integration of BR and GA have been widely documented to play key roles in regulating plant growth, development, and photomorphogenesis. The two most common models of these interactions are the “signaling” model positing a DELLA‐BZR1 interaction (Ross & Quittenden, 2016) and the GA synthesis theory (Jager et al., 2005; Unterholzner et al., 2015). However, the mechanism by which interactions between BRs and GAs mediate the trade‐off between growth and defense in response to P. melonis infection in cucumber remains unclear. Here, we provide evidence that BR signaling fine‐tunes GA biosynthesis via BZR‐mediated transcriptional regulation of the key GA biosynthesis gene gibberellin 20 oxidase 1 (GA20ox1) and that BR‐induced disease resistance is dependent on the interaction with GAs.

RESULTS

BR mediates the resistance of cucumber to P. melonis

To clarify the role of BRs in governing plant resistance to P. melonis, we first characterized the responses of WT and cyp85a1 mutant plants, with and without exogenous EBR treatment, to inoculation with P. melonis. At 3 days after inoculation, seedlings of the cyp85a1 mutant were severely susceptible to P. melonis, with plants exhibiting obvious shrinkage and water soaking with necrotic spots (Figure 1A,B); the disease index reached 69.05% (Figure 1C). However, WT plants displayed only mild disease symptoms with a disease index of 47.41%. Exogenous foliar application of 100 μM EBR was performed to test the potential infection‐suppression effect of EBR. As expected, supplying plants with EBR increased the resistance of cucumber plants to P. melonis infection as the disease index decreased to 34.81% and 33.09% in cyp85a1 and WT plants, respectively, after EBR treatment (Figure 1C).

Figure 1.

Figure 1

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BRs suppressed Phytophthora melonis‐induced disease spreading on cucumber plants 3 days after inoculation.

(A) Phenotypes of P. melonis infected cyp85a1 and WT plants with or without EBR application.

(B) Closeup of visible water‐soaking, brownish lesions, or plant collapse.

(C) Disease incidence in the plants with and without EBR treatment infected with P. melonis.

(D) Invasive hyphae extended on the epidermis and transverse section of hypocotyls stained with lactophenol‐trypan blue.

(E) P. melonis biomass accumulation in the hypocotyls infected with P. melonis.

CH, confined hypha; EBR, 24‐epibrassinolide; IH, invasive hypha; TC, tissue collapse; W, water soaking with necrosis; WT, wild type. Bars = 10 μm. Data in (E) are shown as means ± SD of three biological replicates (n = 9). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

A histological analysis was performed to observe the infection process of P. melonis. At 3 days after inoculation, extensively branched hyphae became evident on the cross‐section and epidermal cells of the hypocotyls of cyp85a1 plants (Figure 1D). However, only a few invasive and confined hyphae developed in untreated WT plants and in both EBR‐treated WT and cyp85a1 plants. RT‐qPCR confirmed that the pathogen biomass in cyp85a1 mutant plants was significantly higher than that in WT plants. The amount of P. melonis DNA per 100 ng genomic DNA in the hypocotyl was 72.65 ng in cyp85a1 plants and was only 45.77 ng in WT plants, with 4.33 and 7.74 ng P. melonis DNA/100 ng genomic DNA in EBR‐treated WT and cyp85a1 plants, respectively (Figure 1E).

BR‐mediated Phytophthora blight resistance is associated with GA biosynthesis

To further explore the regulatory mechanism of BR‐mediated Phytophthora blight resistance, the hypocotyls of cyp85a1 and WT plants with or without P. melonis infection were sampled at two‐leaf stage for comparative RNA‐sequencing analysis 3 days after P. melonis inoculation (Figure S2; Table S3). Based on global transcription changes, the expression of bioactive GA biosynthetic genes was found to be significantly hindered in the cyp85a1 mutant upon P. melonis infection (Figure S3; Tables S4 and S5). The expression of GA20ox3 (CsaV3_6G005990) and gibberellin 3‐beta‐dioxygenase 1 (GA3ox1; CsaV3_7G032870) was all down‐regulated in response to P. melonis infection in cyp85a1 mutant hypocotyls; however, the expression levels of these two genes did not significantly change in infected‐hypocotyls of WT plants. Consequently, the higher expression levels of GA20ox3 and GA3ox1 were observed in infected hypocotyls of WT plants as compared to those of cyp85a1 mutant plants (Figure 2A, WT PM‐infected vs cyp85a1 PM‐infected; Table S5). The expression of KAO (CsaV3_3G049400) was down‐regulated in infected‐hypocotyls of both genotypes upon infection but to a greater extent in cyp85a1 mutant plants. The expression levels of gibberellin 7 oxidase 1 (GA7ox1; CsaV3_1G005350) and GA20ox1 (CsaV3_5G005560) decreased in infected‐hypocotyls of both genotypes upon infection to a similar extent. Moreover, the expression of gibberellin 2 oxidase 1 (GA2ox1; CsaV3_4G007790), which encodes the enzyme that converts bioactive GA4 to inactive GA34, was up‐regulated in infected hypocotyls of both genotypes upon infection, but to a greater level in cyp85a1 mutants than in WT plants. The CYP85A1 mutation did not affect the expression of GA biosynthetic genes in non‐inoculated hypocotyls (Figure 2A, WT vs cyp85a1; Table S5). The expression patterns of the above DEGs were verified using RT‐qPCR (Figure 2B).

Figure 2.

Figure 2

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Transcriptional profiling of Phytophthora melonis‐infected hypocotyls of WT versus cyp85a1 plants 3 days after inoculation revealed the majority of GA biosynthetic genes involved in BR‐mediated phytophthora blight resistance.

(A) Loss of function of CYP85A1 inhibited the expression of GA biosynthetic genes in P. melonis‐infected cucumber hypocotyls. Heatmap colors represent the gene expression (log2fold change) for each comparison (Tables S4 and S5).

(B) Validation of RNA‐seq gene expression using quantitative real time PCR (qRT‐PCR). Data in (B) are shown as means ± SE of three biological replicates (n = 9). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests. cyp85a1 PM infected, P. melonis‐infected cyp85a1; WT PM infected, P. melonis‐infected WT; WT, non‐inoculated WT; cyp85a1, non‐inoculated cyp85a1; WT, wild type.

Next, to confirm the interaction between different BR levels in the plants and growth, disease resistance, and GA biosynthesis, we further transformed the inbred cucumber line CCMC with the construct 35S:CYP85A1 into the pBI121‐GFP vector (Figure S4). Compared to those of the WT CCMC plants, CYP85A1‐OE lines exhibited an enhanced growth rate and improved disease resistance (Figure 3A–C). In addition, the hypocotyl thickness, hypocotyl length, leaf number, and leaf area of CYP85A1‐OE lines were significantly increased in comparison to those of WT plants (Figure 3A; Figure S5). Furthermore, the disease index of OE‐2 and OE‐3 plant lines decreased by 55.87% and 52.92% compared to that of the WT CCMC plants, whereas the EMS‐derived mutant cyp85a1 deficient in BR biosynthesis showed a 52.54% increase in the disease index (Figure 3B–D). With respect to endogenous GAs biosynthesis, GA1 and GA3 levels significantly decreased in OE‐2 plants unpredictably, but there was no change in the levels of these hormones in the EMS‐derived mutant cyp85a1 compared with those of WT plants under the non‐inoculation condition. However, after P. melonis inoculation, GA1 levels were increased in the P. melonis‐infected hypocotyls of all three genotypes compared to those of non‐inoculated plants, although the differences were not statistically significant. Notably, GA3 levels were markedly reduced in the cyp85a1 mutant in response to pathogen infection, but did not change in either the WT or OE‐2 plants (Figure 3E,F). Thus, we speculated that the regulation of Phytophthora blight resistance by BRs might involve fine‐tuning of the endogenous GA3 content.

Figure 3.

Figure 3

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BR‐mediated phytophthora blight resistance is associated with GA biosynthesis.

(A) Phenotypes of cyp85a1, WT and CYP85A1‐OE plants on day 0 before inoculation.

(B) Phenotypes of Phytophthora melonis infected cyp85a1, WT and CYP85A1‐OE plants 3 days after inoculation.

(C) Closeup of visible water‐soaking, brownish lesions, or plant collapse.

(D) Disease incidence in cyp85a1, WT and CYP85A1‐OE plants.

(E) Endogenous GA1 levels.

(F) Endogenous GA3 levels. Data in (E, F) are shown as means ± SE of three biological replicates (n = 9). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

EBR, 24‐epibrassinolide; WT, wild type.

To further verify the involvement of GAs in BRs‐regulated plant growth and disease resistance, we examined the effects of exogenous GA3 pretreatment. Complementing cyp85a1 with exogenous GA3 completely rescued the hypocotyl diameter, leaf number, and leaf area; partly rescued petiole length; and further elongated the first internode length. However, hypocotyl lengthening was not promoted by GA3 treatment. Providing WT plants with exogenous GA3 also substantially promoted plant growth. Supplying plants with both GA3 and EBR exhibited additive effects in promoting the above growth parameters to different extents. Moreover, both the WT and cyp85a1 mutant plants supplemented with exogenous GA3 or EBR had a lower chlorophyll content compared to that of the corresponding plants without chemical treatments. Notably, WT plants had no first internode (Figure 4A; Figure S6). In terms of disease resistance, exogenous GA3 treatment completely rescued the reduced resistance of the cyp85a1 mutant to the level of WT plants, as indicated by a decreased in pathogen biomass accumulation and disease index. GA3 treatment also slightly increased resistance in WT plants. However, co‐treatment of EBR and GA 3 did not confer an additive effect on the level of resistance in either the WT or cyp85a1 mutant plants (Figure 4B–E).

Figure 4.

Figure 4

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EBR and GA3 application suppressed Phytophthora melonis‐induced disease spreading on cucumber plants 3 days after inoculation with P. melonis.

(A) Phenotypes of cyp85a1 and WT plants with or without EBR and GA3 application on day 0 before inoculation.

(B) Phenotypes of P. melonis infected cyp85a1 and WT plants with or without EBR and GA3 application.

(C) Closeup of visible water‐soaking, brownish lesions, or plant collapse of the hypocotyls.

(D) P. melonis biomass assays.

(E) Disease incidence assays.

TC, tissue collapse. Data in (D) are shown as means ± SE of three biological replicates (n = 9). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

EBR, 24‐epibrassinolide; WT, wild type.

To further investigate the role of BR signaling in plant growth and disease resistance, and to clarify the relationship between BRs and GAs in these processes, two homologous transcription factors mediating BR signaling (BZR2 and BZR6) were silenced by VIGS. Transcript levels of BZR2 or BZR6 in the hypocotyls of plants infiltrated with pV190‐BZR2 or pV190‐BZR6 were confirmed to be 54% or 58% lower than those of plants infiltrated with the pV190 vector control, respectively (Figure S7). Silencing of BZR2 or BZR6 reduced hypocotyl thickness, but had no effect on the other growth indices; exogenous application of GA3 significantly increased the hypocotyl thickness, petiole length, and leaf number in BZR2‐ or BZR6‐silenced plants (Figure 5A; Figure S8). Moreover, silencing of BZR6 prevented disease development, and the disease index decreased by 34.48% compared to that of plants infected with the control vector pV190. By contrast, silencing of BZR2 had no effect on resistance. In response to P. melonis infection, both GA1 and GA3 accumulation in BZR6‐silenced plants increased to a greater extent than found in the pV190‐infected and BZR2‐silenced plants. In addition, pre‐treatment with GA3 abolished the enhanced resistance induced by silencing BZR6, with an increase in the disease index from 52.78% to 83.33% (Figure 5B–F). However, GA3 treatment had little effect on the disease resistance of BZR2‐ silenced plants.

Figure 5.

Figure 5

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GA3 application abolished the disease resistance induced by silencing of BZR6 3 days after inoculation with Phytophthora melonis.

(A) Phenotypes of BZR2‐ and BZR6‐silenced plants with or without GA3 application on day 0 before inoculation.

(B) Phenotypes of P. melonis infected BZR2‐ and BZR6‐silenced plants with or without GA3 application.

(C) Closeup of visible water‐soaking, brownish lesions, or plant collapse of the hypocotyls.

(D) Disease incidence assays.

(E) Endogenous GA1 levels.

(F) Endogenous GA3 levels.

TC, tissue collapse; W, water soaking with necrosis. Data in (E, F) are shown as means ± SE of three biological replicates (n = 9). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

BRs regulate GA biosynthesis through the BZR6‐dependent transcriptional regulation of GA20ox1

To verify whether BZR2 or BZR6 has a regulatory effect on the GA biosynthetic gene GA20ox1, the promoter region upstream of GA20ox1 (upstream 1218 bp) was inspected. The promoter contains five E‐box elements (CANNTG), which could be a binding site of the BZR transcription factor (Figure 6A). The yeast‐one‐hybrid assay was performed using five different constructs, each containing one E‐box element. As shown in Figure 6(B) and Figure S9, only the yeast cells that contained the bait vector harboring the GA20ox1 promoter P2 region grew normally on the deficient SD/‐Leu medium when transformed with BZR2‐AD or BZR6‐AD. In addition, the yeast cells harboring both the GA20ox1pro‐bait vector containing the mutated E‐box region of the GA20ox1 promoter P2 and the pGADT7‐BZR2 or pGADT7‐BZR6 vector could not grow on the selective medium. A LUC assay was performed to further visualize the interaction. Examination of N. benthamiana infiltrated with the above constructs using a plant live imaging system showed that the fluorescence intensity was weaker than that of the control (empty vector), clearly demonstrating that BZR2 or BZR6 could bind to the GA20ox1 promoter and consequently suppress GA20ox1 expression (Figure 6C,D). The relative LUC activity assay also showed that the activity of the GA20ox1 promoter was inhibited by 44% or 55% as a result of BZR2 or BZR6 binding, respectively (Figure 6E). Collectively, these results indicated that BZR2 and BZR6 negatively regulate GA20ox1 transcription through direct binding to its promoter in vitro. For comparison, we also performed the yeast‐one‐hybrid assay for GA20ox3, which showed that neither BZR2 nor BZR6 interacts with GA20ox3 (Figure S9).

Figure 6.

Figure 6

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BZR2 and BZR6 directly suppressed the transcription of GA20ox1.

(A) Schematic diagrams of the GA20ox1 promoters.

(B) Yeast one‐hybrid analysis of BZR2 or BZR6 binding to the GA20ox1 promoter P2 region. WT and mutated promoter P2 region were used for analysis. Interaction was determined on synthetic defined (SD) medium lacking Leu in the presence of AbA (150 ng/mL).

(C) Schematic diagram of the effectors, reporters, and internal compositions.

(D) Dual luciferase reporter system assays showed the binding of BZR2 or BZR6 to the GA20ox1 promoter in vivo. The fluorescence intensity weaker than control (empty vector) meant that BZR2 or BZR6 transrepressed the expression of GA20ox1. Label 1: Co‐expression of pGreenΙΙ 0800‐Luc vector and pGreenII 62‐SK vector. Label 2: Co‐expression of proGA20ox1::LUC and pGreenII 62‐SK vector. Label 3: Co‐expression of pGreenΙΙ 0800‐Luc vector and 35S::BZR2/6. Label 4: Co‐expression of proGA20ox1::LUC and 35S::BZR2/6.

(E) Dual luciferase assay for the regulatory effect of BZR2 or BZR6 on the expression of GA20ox1. The ratio of LUC/REN of the SK plus promoter was set to 1 for normalization. Data in (E) are shown as means ± SE of three biological replicates (n = 9). Asterisks indicated significant differences (P < 0.05) according to Student's t‐test.

GA biosynthesis is necessary for BR‐mediated Phytophthora blight resistance in cucumber

To investigate whether GA biosynthesis is essential for the observed EBR‐enhanced plant growth and disease resistance, two GA biosynthesis genes, GA20ox1 and KAO, were silenced by VIGS and then EBR was applied exogenously to the gene‐silenced plants for extensive analysis. The transcript levels of GA20ox1 or KAO in the hypocotyls of plants infiltrated with pV190‐GA20ox1 or pV190‐KAO were 82% or 57% lower than those of plants infiltrated with the pV190 vector control, respectively. Under non‐inoculation conditions, silencing of GA20ox1 or KAO significantly inhibited plant growth; the plants exhibited delayed growth and dwarfism. The EBR‐induced petiole lengthening was hindered to different extents by silencing of GA20ox1 or KAO (Figure 7A; Figure S10). Furthermore, the EBR‐induced disease resistance was blocked by silencing GA20ox1 or KAO, as indicated by the increase in the disease index and P. melonis biomass. The repression of disease resistance was greater in GA20ox1‐silenced plants than in KAO‐silenced plants (Figure 7B–E).

Figure 7.

Figure 7

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EBR‐induced disease resistance was blocked by silencing of GA20ox1 or KAO 3 days after inoculation with Phytophthora melonis.

(A) Phenotypes of GA20ox1‐ and KAO‐silenced plants with or without EBR application on day 0 before inoculation.

(B) Phenotypes of P. melonis infected GA20ox1‐ and KAO‐silenced plants with or without EBR application.

(C) Closeup of visible water‐soaking, brownish lesions, or plant collapse of the hypocotyls.

(D) Disease incidence assays.

(E) P. melonis biomass accumulation in the plants infected with P. melonis. TC, tissue collapse.

(F) A proposed working model of BR‐GA crosstalk in the regulation of cucumber growth and P. melonis resistance. Data in (E) are shown as means ± SE of three biological replicates (n = 9). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

BR, brassinosteroids; EBR, 24‐epibrassinolide.

DISCUSSION

The continuous coevolutionary struggle of hosts and pathogens is complex. Plant hormones are small‐molecule signals, which not only orchestrate fundamental developmental cues but also convey environmental inputs and induce an adaptive response to abiotic and biotic stresses. Plant hormones, such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), are well‐established to play an important role in plant immunity (Wang, Tyler et al., 2019). In this study, we found that GA biosynthesis is necessary for the BRs‐mediated increased resistance of cucumber plants to P. melonis and uncovered a regulatory module of BZR6‐GA20ox1.

Studies in plant–oomycete interactions elucidated the complex roles and action mechanisms of BRs in mediating the plant disease response using BR biosynthesis and signaling mutants along with exogenous treatment of active BRs (de Bruyne et al., 2014; Lozano‐Durán et al., 2013; Yu et al., 2018). Exogenous EBR treatment increased the resistance of soybean plants to P. sojae (Wang et al., 2020), and we also previously found similar results in cucumber plants against P. melonis (Ren et al., 2020). Silencing of the BR receptor gene SERK3/BAK1 in potato increased the susceptibility of the plants to P. infestans, which was accompanied by reduced expression of the BR marker genes StSTDH, StEXP8, StCAB50, and StCHL1 (Wang, He et al., 2019). In tomato plants, the disease severity of curl3, a mutant that is insensitive to BR, was significantly higher than that of WT plants after inoculation with P. infestans (Dallagnol et al., 2023). In addition, overexpression of MdBES1 or MdMYB88 in apple increased the disease resistance against V. mali infection. MdBES1 could directly bind to the MdMYB88 promoter, and MdMYB88 promoted BR biosynthesis by upregulating the expression of BR biosynthesis genes (Liu et al., 2021). In this study, both P. melonis inoculation of plants varying in BR biosynthesis capacity and exogenous application of EBR uncovered a positive role of BRs in the disease response (Figures 1 and 3), suggesting that BRs function as a resistance factor to enhance the resistance of cucumber plants to P. melonis. However, BZR6, a key transcription factor in the BR signaling pathway, was found to negatively regulate the resistance to P. melonis (Figure 5).

The mechanisms by which GA plays a role in regulating disease resistance are complex. The DELLA protein SLENDER RICE1 (SLR1) showed a resistance‐promoting effect against hemibiotroph rice pathogens by boosting SA‐ and JA‐mediated defense responses (de Vleesschauwer et al., 2016). Knockout of the Elongated uppermost internode (Eui) gene, encoding a GA deactivating enzyme, or exogenous application of GA3 reduced disease resistance to Xanthomonas oryzae pv. oyrzae and Magnaporthe oryzae, whereas Eui overexpression increased disease resistance (Yang et al., 2008). By contrast, inoculation of GA20ox3‐overexpression and RNA‐interference lines with the rice pathogens M. oryzae and X. oryzae pv. oryzae revealed a positive role of GA in disease resistance (Qin et al., 2013). Exogenous application of GA3 could restore the normal growth phenotypes of dwarf rice plants infected by Rice dwarf virus (Zhu et al., 2005). In addition, GA was found to mitigate the damage caused by citrus Huanglongbing (i.e., citrus greening disease) through inhibiting H2O2 production and cell death in phloem tissues (Ma et al., 2022). In the present study, knockdown of KAO or GA20ox1 significantly reduced the resistance of cucumber to P. melonis (Figure 7). The effects of exogenously applied GA3 on the disease resistance of cucumber plants varied according to the genotype of the plant. In the cyp85a1 mutant with a reduced GA3 level upon infection, GA3 application significantly enhanced resistance (Figures 3 and 4). However, for the WT, pV190‐infected, and BZR2‐silenced plants with no change in GA3 level upon infection, exogenous GA3 treatment only increased the resistance of WT and pV190‐infected plants, but had no influence on the resistance of BZR2‐silenced plants (Figures 4 and 5). Moreover, exogenous application of GA3 to BZR6‐silenced plants with a relatively increased endogenous GA3 level upon infection counteracted the resistance induced by silencing BZR6 (Figure 5). Hence, we concluded that GA‐induced Phytophthora blight resistance in cucumber plants not only relies on modulation of the endogenous GA3 content, but also involves the interaction of GA with the BR signaling pathway. Consequently, GA3 over‐production might trigger feedback regulation of the BZR2/BZR6‐mediated resistance response.

Several lines of evidence indicate that BR/GA cross‐talk putatively plays a crucial role in the BR‐mediated resistance to P. melonis. First, transcriptional profiling of WT and mutant cyp85a1 cucumber plants revealed the potential role of GA biosynthesis in BR‐mediated Phytophthora blight resistance (Figure 2; Figure S3). Second, functional analysis of CYP85A1, BZRs, GA20ox1, and KAO confirmed that GA biosynthesis is necessary for BRs‐induced disease resistance. The BR‐deficient cyp85a1 mutant plants showed more severe disease symptoms than the WT plants, accompanied by a reduced endogenous GA3 content after pathogen infection (Figure 3F), whereas external foliar application of GA3 to the cyp85a1 mutant completely rescued the resistance to the WT level (Figure 4). In addition, transient knock down of BZR6 increased disease resistance and simultaneously promoted the accumulation of endogenous GA1 and GA3 in cucumber after P. melonis infection compared with those of WT plants (Figure 5E,F). More importantly, the disease resistance conferred by exogenous EBR was abolished by silencing GA20ox1 or KAO (Figure 7). Third, the results of yeast one‐hybrid and luciferase assays suggested that BZR6 directly suppressed the transcription of GA20ox1 through binding to its promoter (Figure 6). However, CYP85A1‐OE plants showed higher disease resistance than WT plants, but had lower GA1 and GA3 levels (Figure 3E,F), and the increased disease resistance induced by silencing BZR6 was abolished by exogenous GA3 treatment (Figure 5). Co‐application of EBR and GA3 also did not have an additive effect on disease resistance compared to the application of either hormone alone in both WT and cyp85a1 plants (Figure 4). Together, these results point to a complex interaction of BRs and GA3 in triggering the plant disease‐resistance response, suggesting that the fine‐tuning of endogenous GA content might be required for the BR‐induced resistance of cucumber plants to P. melonis. Nevertheless, our results are somewhat inconsistent with the findings of a previous study in rice showing that BR antagonized GA‐directed root immunity via indirectly stabilizing the DELLA protein and the central GA repressor SLR1 (de Vleesschauwer et al., 2012).

The ontogenic or age‐related resistance (ARR) phenomenon to Phytophthora fruit rot has been documented in Cucurbitaceae. The young fruits are extremely susceptible to Phytophthora infection but become resistant throughout growth and development (Alzohairy et al., 2020; Ando et al., 2009; Mansfeld et al., 2017, 2020). Although such an ARR pattern to Phytophthora root and crown rot has not been reported in Cucurbitaceae, it has been widely documented in Solanaceae vegetable crops including tomato (Shah et al., 2015), potato (Mutty & Hossenkhan, 2008) and pepper (Jeun & Hwang, 1991). In this study, the plants with reduced levels of endogenous BRs or GAs through knockout of the biosynthesis gene CYP85A1, KAO, or GA20ox1, resulting in delayed growth compared with that of WT plants, were also more susceptible to P. melonis (Figures 3 and 7; Figures S5 and S10). In addition, CYP85A1 overexpression promoted plant growth and increased plant disease resistance (Figure 3; Figure S5). These results seem to support a mechanism of BR/GA‐mediated growth‐related resistance against P. melonis infection. However, no correlation between growth rate and disease resistance in BZR6‐ or BZR2‐silenced plants was observed, regardless of with or without GA3 treatment, which does not provide support for the ARR theory (Figure 5; Figure S8). We also did not find an additive promotion effect of EBR and GA3 on plant growth in WT or cyp85a1 mutant plants with respect to pathogen resistance (Figure 4; Figure S6). Hence, a trade‐off between plant disease resistance and growth is required in the co‐evolutionary struggle of hosts and pathogens. BRs‐regulated disease resistance is not simply ascribed to a mechanism of growth‐related resistance, suggesting that the resistance mechanism is more complex.

CONCLUSION

Our results provide evidence that BRs positively regulate the resistance of cucumber plants to the pathogen P. melonis and reveal a multifaceted regulation mechanism of BR/GA cooperation on growth‐defense trade‐offs. These findings can help to inform the development of a valuable strategy for cucumber blight control in production.

EXPERIMENTAL PROCEDURES

Plant materials

The wild‐type (WT) and cyp85a1 mutant cucumber (Cucumis sativus L.) lines were kindly provided by Dr. Yuhong Li (Northwest A & F University). The WT line ‘Changchun mici’ (CCMC) is a North China fresh type inbred line, which was used for virus‐induced gene silencing (VIGS) and genetic transformation. The BR‐deficient mutant cyp85a1 (encoding BR‐C6‐oxidase in the BR biosynthesis pathway) is an ethyl methanesulfonate (EMS)‐derived mutant isogenic CCMC cucumber line with lower BR levels relative to those of the WT. Loss of function of CYP85A1 results in an extremely dwarf phenotype with practically no internodes, and small, dark green, and severely wrinkled leaves. Foliar spraying of 100 μM EBR partially rescues the mutant phenotypes (Wang et al., 2017).

Treatments with exogenous plant hormones

We examined the effects of EBR and GA3 on the promotion/suppression of growth and disease resistance of cyp85a1 mutant, WT CCMC plants, and WT plants with silencing of GA biosynthetic genes GA20ox1 and ent‐kaurenoic acid oxidase (KAO) and BR signaling genes (BZR2 and BZR6) individually.

The seedlings of cyp85a1 mutant plants were sprayed with 5 mL of 100 μM EBR (Sigma‐Aldrich, Germany; catalog #E1641) and 5 mL of 100 μM GA3 (Solarbio, China; catalog #G8040) per plant every day when the cotyledons were fully expanded. Notably, the cyp85a1 seedlings emerged from soil surface 1 week later than WT seedlings, due to the extremely short hypocotyl and practically no epicotyl in the mutant. To confirm restoration of the phenotype in cyp85a1 via EBR administration, plant growth was evaluated 13 days after EBR treatments in WT and 6 days after treatment in cyp85a1, and then the plants were inoculated with P. melonis the following day. For evaluation of restoration/suppression of the phenotype in the cyp85a1 by GA3, morphological traits and disease resistance were determined 15 days after GA3 treatments in WT plants and 8 days after treatment in cyp85a1 plants, and then the plants were inoculated with P. melonis the following day.

The seedlings of GA20ox1‐ or KAO‐silenced plants at the two‐leaf stage were sprayed with 10 mL of 100 μM EBR per plant every day, whereas the seedlings of BZR2‐ or BZR6‐silenced plants at the two‐leaf stage were sprayed with 10 mL of 100 μM GA3 per plant every day. Plant growth was evaluated 5 days after chemical treatments, followed by inoculation with P. melonis. The stock solutions of EBR and GA3 were dissolved in ethanol and diluted with distilled water.

Plant growth and inoculations

Cucumber plants were grown as described by Ren et al. (2020). In brief, the growth chamber (Ningbo, model RXZ 500‐D with fluorescent lamps) was programmed at 26°C during the day and 20°C at night under a 16‐h photoperiod with a 24 000‐lx light intensity, and the relative humidity was set to 70%. For the cyp85a1 mutant plants treated with plant hormones, inoculation with P. melonis was initiated when the cyp85a1 mutant plants developed two to three true leaves. For the GA20ox1‐, KAO‐, BZR2‐ and BZR6‐silenced plants, P. melonis inoculation was initiated 5 days after plant hormones treatments. Considering that cyp85a1 mutant plants present a dwarf hypocotyl phenotype, we supported the leaves from the soil with a 10‐μL pipette tip to avoid direct contact of the leaves with zoospores during inoculation (Figure S1).

A local isolate of P. melonis, TARIp211155 (accession no. MK386561.1) collected from cucumber in South China, was used for inoculations. Zoospore suspensions were prepared according to the method as described by Xu et al. (2015). The plants were inoculated by adding 20 mL of the zoospore suspensions (containing 1 × 104 zoospores per milliliter) to the substrate near the stem base. Incubation was performed at 27°C in the dark for 24 h, followed by the normal diurnal cycle culture conditions mentioned above. Disease progression was observed 3 days after inoculation and scored based on a scale ranging from 0 to 4 as follows: 0, no symptoms; (1) barely water‐soaking or brownish lesions appearing on the crown; (2) water‐soaking or brownish lesions with slight constriction of the crown; (3) constrited/girdling crown progressed to become larger than 25% of the whole hypocotyl; and (4) death of the plant (Ren et al., 2020). The disease index (DI) was calculated from at least 10 individual plants per replicate according to the following equation:

DI=∑disease scale value*number of plantsateach disease scalemaximum disease scale value*total number of plants surveyed*100%

In vivo analyses of P. melonis infection

For the visualization of fungal structures, the inoculated hypocotyl epidermis and transverse sections of inoculated hypocotyls were stained with lactophenol‐trypan blue as described previously (Tao, 2012) 3 days after inoculation. The transverse section of the hypocotyl (10 mm long starting from the basal end) was cut into small pieces with a razor blade.

DNA extraction and fungal biomass quantification

DNA extraction and detection of P. melonis from cucumber hypocotyls were performed according to the methods of Wang et al. (2007). Briefly, fungal biomass accumulation was quantitatively assessed by amplifying the internal transcribed spacer region of the nuclear ribosomal DNA of P. melonis (Table S1). The calibration curve was constructed by plotting the logarithm of a known concentration (a 10‐fold dilution series from 100 ng to 10 pg in a reaction volume of 25 μL) of DNA from P. melonis against threshold cycle (C t) values. The regression equation was C t = −3.6327 log[DNA] + 29.028, with a coefficient of determination (R 2) of 0.9997.

RNA‐sequencing and differential gene expression analysis

When the seedlings developed two true leaves, the inoculated and non‐inoculated hypocotyls of cyp85a1 mutant and WT plants were sampled for RNA‐sequencing analysis 3 days after P. melonis inoculation. RNA extraction and cDNA library construction of 12 cucumber hypocotyls were conducted by Biomarker Technologies (Beijing, China) according to standard procedures. Sequencing of all cDNA libraries was performed on an Illumina HiSeq2500 sequencer with approximately 50 million paired‐end strand‐specific reads produced per sample. Raw data (raw reads) in fastq format were processed through in‐house Perl scripts. Clean reads were obtained by removing reads containing adapters or poly‐N sequences and low‐quality reads from the raw data. A total of 117.28 Gb clean reads were generated, and the percentage of Q30 bases in each sample was greater than 94.06% (Table S2). Clean reads were aligned to the cucumber (Chinese Long) v3 reference genome using Hisat2 software and the mapped reads were quantified using StringTie software. Gene expression levels were estimated by the fragments per kilobase of transcript per million fragments mapped reads (FPKM) method. Differential expression analysis of two groups (Table S3; WT vs. cyp85a1; non‐inoculated vs. inoculated cyp85a1; non‐inoculated vs. inoculated WT; inoculated WT vs. inoculated cyp85a1) was performed using the DESeq2 package. Genes with a difference in expression level at an adjusted P‐value < 0.05 and fold Change ≥4 were considered to be differentially expressed genes (DEGs).

Gene Ontology (GO) enrichment analysis of the DEGs was implemented by the GOseq R packages based on the Wallenius non‐central hyper‐geometric distribution (Young et al., 2010). Venn Diagrams were generated using TBtools software (Chen et al., 2020). Enrichment analysis was performed using KOBAS (http://kobas.cbi.pku.edu.cn/genelist/) and the results were visualized with R tools (Bu et al., 2021).

VIGS

Gene functions were analyzed using the VIGS system derived from the cucumber green mottle mosaic virus (Liu et al., 2020). Four target genes, BZR2, BZR6, GA20ox1, and KAO, were silenced using recombinant virus pV190 VIGS vectors as previously described (Liao et al., 2019) to analyze their functions during P. melonis infection in cucumber plants. The primers are listed in Table S1. In brief, the constructed plasmids were transformed into Agrobacterium tumefaciens strain GV3101. The VIGS inoculum was first infiltrated into the sprouts of the inbred cucumber line CCMC according to the methods of Liao et al. (2019), and then infiltrated into the cotyledons at the cotyledon stage to enhance the infection efficiency. Cucumber phytoene desaturase (PDS; encoded by CsaV3_4G002690) was used as a positive control to monitor the silencing efficiency based on the presence or absence of photo‐bleaching phenotypes. Plants infiltrated with an Agrobacterium culture carrying the empty pV190 VIGS vector served as controls. At the two‐leaf stage, the newly emerged leaves from the pV190‐PDS‐inoculated plants showed highly uniform photobleaching symptoms. The hypocotyls were sampled to quantify the silencing effect by determining the expression of targeted genes using reverse‐transcription quantitative polymerase chain reaction (RT‐qPCR). The plants that showed less than 50% transcript levels relative to those of control plants were foliarly applied with the plant hormone every day for 5 days, followed by inoculation with P. melonis.

Cucumber transformation

To obtain cucumber plants overexpressing CYP85A1 (CYP85A1‐OE), the full‐length coding sequence of CYP85A1 was cloned into the pBI121‐GFP vector to generate the overexpression constructs. The CYP85A1‐OE constructs were introduced into A. tumefaciens via chemical transformation (Wise et al., 2006) and then transformed into the inbred cucumber line CCMC. Vacuum infiltration was used to enhance Agrobacterium infection. Green fluorescent protein (GFP) was used as a reporter in the selection of transgenic shoots (Nanasato et al., 2013; Zhang et al., 2017). GFP screening, PCR, sequencing, and western immunoblotting were used to characterize transgenic lines. The primers used for vector construction and transformation are listed in Table S1. Protein extraction and immunoblots analysis were performed according to the methods described by Yu et al. (2012). Total proteins isolated from the transgenic cucumber plants were assessed using an anti‐GFP tagged primary monoclonal antibody (Agrisera, Sweden; catalog #AS18 4175).

Quantification of endogenous GA

For GA quantification, 1 g of the cucumber hypocotyl was sampled for each of three biological replicates. GA1 and GA3 levels were measured by ultra‐high‐performance liquid chromatography‐mass spectroscopy (Xevo TQD; Waters, Milford, MA, USA) coupled with an Alliance e2695 system (Waters) as described by Xin et al. (2020).

Yeast one‐hybrid assay

The full‐length cDNA of BZRs was linked to the pGADT7 vector (Clontech‐Takara Bio) as a bait. The promoter fragment of GA20ox1 and GA20ox3 (containing the binding site of BZR1/BES1) was linked to the pAbAi vector as a prey. To obtain the mutant E‐box, the CAAGTG sequence was mutated to CAAGCC using the Mut Express MultiS Fast Mutagenesis Kit V2 (Vazyme, catalog # C215, Nanjing, China). The primers used to construct the vector are listed in Table S1. The pAbAi‐GA20ox1 and GA20ox3 construct were linearized and transferred into yeast cells and cultured on selective synthetic‐defined (SD) medium lacking uracil. Next, recipient cells containing GA20ox1 and GA20ox3 promoter fragments were prepared using the Yeastmaker™ Yeast Transformation System 2 (Clontech‐Takara Bio, catalog #630439, Japan), and BZRs were co‐transformed into recipient cells and grown on the SD/‐Leu medium (AbA Clontech‐Takara Bio, catalog #630466, Japan).

Dual‐luciferase reporter assays

The 2000‐bp promoter fragment containing the GA20ox1 binding site was cloned into the pGreenΙΙ 0800‐Luc vector (Biovector, Beijing, China) to generate the reporter (proGA20ox1::LUC). The coding sequence of BZR2 or BZR6 was cloned into the pGreenII 62‐SK vector (Biovector, Beijing, China) to generate the effector (35S::BZR2/6). The effector and reporter plasmids were then co‐injected into the leaves of N. benthamiana. At 48 h after inoculation, fluorescein potassium salt (Solarbio, catalog #IL2330, China) was uniformly applied to the abaxial surface of the leaf, and the fluorescence signal was detected 10 min later using a plant live imaging system (Berthold/NightSHADE LB985) (Gao et al., 2023). Luciferase activities were measured on a Luminoskan Ascent microplate luminometer (Thermo, Waltham, MA, USA) using the LUC gene test kit (Promega, Madison, WI, USA) (Hellens et al., 2005).

RNA isolation and gene expression analysis

Total RNA from samples of the WT and mutant plant hypocotyls was extracted using an RNA extraction kit (Tiangen, catalog # DP360, Beijing, China) following the manufacturer's protocol. Subsequently, 1 μg of total RNA was reverse‐transcribed into cDNA using PrimerScript™ RT reagent kit with gDNA Eraser (Clontech‐Takara Bio, catalog # RR047A, Japan). qPCR was conducted on a CFX Connect Real‐Time PCR Detection System (Bio‐Rad, USA) with ChamQ SYBR qPCR Master Mix (Vazyme, catalog # Q341‐02, China). The primers are listed in Table S1. The expression levels of target genes were normalized against the level of the ubiquitin extension protein (UBI‐ep) gene (CsaV3_5G031430) as the reference and calculated using the 2−ΔΔCT method (Wan et al., 2010).

Statistical analysis

Data are presented as mean ± standard error (SE). Growth and disease rating determinations were performed with at least 10 individual plants per biological replicate. Determinations of the other parameters were performed with three independent tissue pools per biological replicate, each tissue pool consisting of three to six plants. All data were statistically analyzed with SAS software (SAS Institute Inc., Cary, NC, USA) using one‐way analysis of variance (ANOVA) and Duncan's multiple range test and assessed at the 0.05 level of significance.

AUTHOR CONTRIBUTIONS

YK, XY and MZ conceived the original research plan; Material preparation, data collection and analysis were performed by ZJ, CM, XN and GP. The first draft of the manuscript was written by YK and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare that there is no conflict of interest regarding the publication of this article.

Supporting information

Figure S1. Pipette tips were used to support plant leaves to prevent direct contamination of leaves by Phytophthora melonis.

Figure S2. (A) An upset plot showing all significantly differentially expressed genes (adjusted P < 0.05, fold change >4) and the intersecting datasets in the hypocotyls of WT versus cyp85a1 plants 3 days after inoculation. (B) Principal component analysis of the transcriptome data. cyp85a1 PM infected, Phytophthora melonis‐infected cyp85a1; WT PM infected, P. melonis‐infected WT; WT, non‐inoculated WT; cyp85a1, non‐inoculated cyp85a1.

Figure S3. Enriched functional subcategories of up‐ (A) and down‐regulated (B) transcripts (adjusted P‐value <0.05) in the hypocotyls of WT versus cyp85a1 plants 3 days after inoculation. Circles' size represents the number of genes (log10) for each functional subcategory. Complete dataset in Table S4. Enrich factor = (differential expressed gene number in a pathway/total differential expressed gene number)/(gene number in a pathway in the database/total gene number in the database). cyp85a1 PM infected, Phytophthora melonis‐infected cyp85a1; WT PM infected, P. melonis‐infected WT; WT, non‐inoculated WT; cyp85a1, non‐inoculated cyp85a1.

Figure S4. (A) Relative CYP85A1 expression levels in different transgenic cucumber lines. (B) Immunoblot analysis to identify CYP85A1 transgenic cucumber lines with an anti‐GFP antibody. Ponceau staining of Rubisco was used as the loading control. Data in (A) were shown as means ± SE of three biological replicates (n = 9). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

Figure S5. The growth characteristics of cyp85a1, WT and CYP85A1‐OE plants under the non‐inoculation condition with Phytophthora melonis. Data were shown as means ± SE of three biological replicates (n = 45). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

Figure S6. The effect of EBR and GA3 application on the growth of cyp85a1 and WT plants under the non‐inoculation condition with P. melonis. Data were shown as means ± SE of three biological replicates (n = 45). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

Figure S7. The leaf bleaching phenotype was observed 14 (A) and 21 days (B) after cotyledons infiltration in pV190‐PDS plants. (C, D) Silencing efficiencies of individual genes through virus‐induced gene silencing (VIGS) in cucumber hypocotyls were determined on day 0 before hormone treatment through RT‐qPCR. Data were shown as means ± SE of three biological replicates (n = 9). Asterisks indicated significant differences (P < 0.05) according to Student's t‐test.

Figure S8. The effect of GA3 application on the growth of BZR2‐ and BZR6‐silenced plants under the non‐inoculation condition with Phytophthora melonis. Data were shown as means ± SE of three biological replicates (n = 45). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

Figure S9. Yeast one‐hybrid analysis of BZR2 or BZR6 binding to the GA20ox1 and GA20ox3 promoters. (A) Schematic diagrams of the GA20ox1 promoters. (B) Schematic diagrams of the GA20ox3 promoters. (C) BZR2 or BZR6 did not bind to GA20ox1 promoter P1, P3, P4, P5 regions. (D) BZR2 or BZR6 did not bind to the promoters of GA20ox3. Interaction was determined on synthetic defined (SD) medium lacking Leu in the presence of AbA (150 ng/mL).

Figure S10. The effect of EBR application on the growth of GA20ox1‐ and KAO‐silenced plants under the non‐inoculation condition with Phytophthora melonis. Data were shown as means ± SE of three biological replicates (n = 30). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

TPJ-119-1353-s002.pdf (3.2MB, pdf)

Table S1. List of primers used in this study.

Table S2. Overview of sequencing quality and alignments. cyp85a1 PM infected, Phytophthora melonis‐infected cyp85a1; WT PM infected, P. melonis‐infected WT; WT, non‐inoculated WT; cyp85a1, non‐inoculated cyp85a1.

Table S3. List of Cucumis sativus differentially expressed genes comparing WT and cyp85a1 hypocotyls infected with or without Phytophthora melonis. cyp85a1 PM infected, P. melonis‐infected cyp85a1; WT PM infected, P. melonis‐infected WT; WT, non‐inoculated WT; cyp85a1, non‐inoculated cyp85a1.

Table S4. Functional enrichment analysis of differentially expressed transcripts in response to Phytophthora melonis infection and CYP85A1 mutation. Functional classification from Bu et al., Nucleic Acids Research 2021. cyp85a1 PM infected, P. melonis‐infected cyp85a1; WT PM infected, P. melonis‐infected WT; WT, non‐inoculated WT; cyp85a1, non‐inoculated cyp85a1.

Table S5. List of differentially expressed genes involved in GA biosynthesis in the hypocotyls of WT versus cyp85a1 plants 3 days after Phytophthora melonis. cyp85a1 PM infected, P. melonis‐infected cyp85a1; WT PM infected, P. melonis‐infected WT; WT, non‐inoculated WT; cyp85a1, non‐inoculated cyp85a1.

TPJ-119-1353-s001.xlsx (6.2MB, xlsx)

ACKNOWLEDGEMENTS

This study was funded by National Natural Science Foundation of China (31872090).

Contributor Information

Xian Yang, Email: yangxian@scau.edu.cn.

Min Zhong, Email: zhongmin@scau.edu.cn.

DATA AVAILABILITY STATEMENT

The raw RNA‐Seq reads have been deposited in the National Center for Biotechnology Information BioProject database (http://www.ncbi.nlm.nih.gov/bioproject) with ID PRJNA858502.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Pipette tips were used to support plant leaves to prevent direct contamination of leaves by Phytophthora melonis.

Figure S2. (A) An upset plot showing all significantly differentially expressed genes (adjusted P < 0.05, fold change >4) and the intersecting datasets in the hypocotyls of WT versus cyp85a1 plants 3 days after inoculation. (B) Principal component analysis of the transcriptome data. cyp85a1 PM infected, Phytophthora melonis‐infected cyp85a1; WT PM infected, P. melonis‐infected WT; WT, non‐inoculated WT; cyp85a1, non‐inoculated cyp85a1.

Figure S3. Enriched functional subcategories of up‐ (A) and down‐regulated (B) transcripts (adjusted P‐value <0.05) in the hypocotyls of WT versus cyp85a1 plants 3 days after inoculation. Circles' size represents the number of genes (log10) for each functional subcategory. Complete dataset in Table S4. Enrich factor = (differential expressed gene number in a pathway/total differential expressed gene number)/(gene number in a pathway in the database/total gene number in the database). cyp85a1 PM infected, Phytophthora melonis‐infected cyp85a1; WT PM infected, P. melonis‐infected WT; WT, non‐inoculated WT; cyp85a1, non‐inoculated cyp85a1.

Figure S4. (A) Relative CYP85A1 expression levels in different transgenic cucumber lines. (B) Immunoblot analysis to identify CYP85A1 transgenic cucumber lines with an anti‐GFP antibody. Ponceau staining of Rubisco was used as the loading control. Data in (A) were shown as means ± SE of three biological replicates (n = 9). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

Figure S5. The growth characteristics of cyp85a1, WT and CYP85A1‐OE plants under the non‐inoculation condition with Phytophthora melonis. Data were shown as means ± SE of three biological replicates (n = 45). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

Figure S6. The effect of EBR and GA3 application on the growth of cyp85a1 and WT plants under the non‐inoculation condition with P. melonis. Data were shown as means ± SE of three biological replicates (n = 45). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

Figure S7. The leaf bleaching phenotype was observed 14 (A) and 21 days (B) after cotyledons infiltration in pV190‐PDS plants. (C, D) Silencing efficiencies of individual genes through virus‐induced gene silencing (VIGS) in cucumber hypocotyls were determined on day 0 before hormone treatment through RT‐qPCR. Data were shown as means ± SE of three biological replicates (n = 9). Asterisks indicated significant differences (P < 0.05) according to Student's t‐test.

Figure S8. The effect of GA3 application on the growth of BZR2‐ and BZR6‐silenced plants under the non‐inoculation condition with Phytophthora melonis. Data were shown as means ± SE of three biological replicates (n = 45). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

Figure S9. Yeast one‐hybrid analysis of BZR2 or BZR6 binding to the GA20ox1 and GA20ox3 promoters. (A) Schematic diagrams of the GA20ox1 promoters. (B) Schematic diagrams of the GA20ox3 promoters. (C) BZR2 or BZR6 did not bind to GA20ox1 promoter P1, P3, P4, P5 regions. (D) BZR2 or BZR6 did not bind to the promoters of GA20ox3. Interaction was determined on synthetic defined (SD) medium lacking Leu in the presence of AbA (150 ng/mL).

Figure S10. The effect of EBR application on the growth of GA20ox1‐ and KAO‐silenced plants under the non‐inoculation condition with Phytophthora melonis. Data were shown as means ± SE of three biological replicates (n = 30). Different letters indicated significant differences (P < 0.05) according to Duncan's multiple range tests.

TPJ-119-1353-s002.pdf (3.2MB, pdf)

Table S1. List of primers used in this study.

Table S2. Overview of sequencing quality and alignments. cyp85a1 PM infected, Phytophthora melonis‐infected cyp85a1; WT PM infected, P. melonis‐infected WT; WT, non‐inoculated WT; cyp85a1, non‐inoculated cyp85a1.

Table S3. List of Cucumis sativus differentially expressed genes comparing WT and cyp85a1 hypocotyls infected with or without Phytophthora melonis. cyp85a1 PM infected, P. melonis‐infected cyp85a1; WT PM infected, P. melonis‐infected WT; WT, non‐inoculated WT; cyp85a1, non‐inoculated cyp85a1.

Table S4. Functional enrichment analysis of differentially expressed transcripts in response to Phytophthora melonis infection and CYP85A1 mutation. Functional classification from Bu et al., Nucleic Acids Research 2021. cyp85a1 PM infected, P. melonis‐infected cyp85a1; WT PM infected, P. melonis‐infected WT; WT, non‐inoculated WT; cyp85a1, non‐inoculated cyp85a1.

Table S5. List of differentially expressed genes involved in GA biosynthesis in the hypocotyls of WT versus cyp85a1 plants 3 days after Phytophthora melonis. cyp85a1 PM infected, P. melonis‐infected cyp85a1; WT PM infected, P. melonis‐infected WT; WT, non‐inoculated WT; cyp85a1, non‐inoculated cyp85a1.

TPJ-119-1353-s001.xlsx (6.2MB, xlsx)

Data Availability Statement

The raw RNA‐Seq reads have been deposited in the National Center for Biotechnology Information BioProject database (http://www.ncbi.nlm.nih.gov/bioproject) with ID PRJNA858502.

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