Salicylic acid-related cotton (Gossypium arboreum) ribosomal protein GaRPL18 contributes to resistance to Verticillium dahliae
- Qian Gong†1,
- Zhaoen Yang†1,
- Xiaoqian Wang1,
- Hamama Islam Butt1,
- Eryong Chen1,
- Shoupu He1,
- Chaojun Zhang1,
- Xueyan Zhang1 and
- Fuguang Li1Email authorView ORCID ID profile
© The Author(s). 2017
Received: 13 October 2016
Accepted: 24 February 2017
Published: 3 March 2017
Verticillium dahliae is a phytopathogenic fungal pathogen that causes vascular wilt diseases responsible for considerable decreases in cotton yields. The complex mechanism underlying cotton resistance to Verticillium wilt remains uncharacterized. Identifying an endogenous resistance gene may be useful for controlling this disease.
We cloned the ribosomal protein L18 (GaRPL18) gene, which mediates resistance to Verticillium wilt, from a wilt-resistant cotton species (Gossypium arboreum). We then characterized the function of this gene in cotton and Arabidopsis thaliana plants. GaRPL18 encodes a 60S ribosomal protein subunit important for intracellular protein biosynthesis. However, previous studies revealed that some ribosomal proteins are also inhibitory toward oncogenesis and congenital diseases in humans and play a role in plant disease defense. Here, we observed that V. dahliae infections induce GaRPL18 expression. Furthermore, we determined that the GaRPL18 expression pattern is consistent with the disease resistance level of different cotton varieties. GaRPL18 expression is upregulated by salicylic acid (SA) treatments, suggesting the involvement of GaRPL18 in the SA signal transduction pathway. Virus-induced gene silencing technology was used to determine whether the GaRPL18 expression level influences cotton disease resistance. Wilt-resistant cotton species in which GaRPL18 was silenced became more susceptible to V. dahliae than the control plants because of a significant decrease in the abundance of immune-related molecules. We also transformed A. thaliana ecotype Columbia (Col-0) plants with GaRPL18 according to the floral dip method. The plants overexpressing GaRPL18 were more resistant to V. dahliae infections than the wild-type Col-0 plants. The enhanced resistance of transgenic A. thaliana plants to V. dahliae is likely mediated by the SA pathway.
Our findings provide new insights into the role of GaRPL18, indicating that it plays a crucial role in resistance to cotton “cancer”, also known as Verticillium wilt, mainly regulated by an SA-related signaling pathway mechanism.
KeywordsCotton Verticillium wilt Resistance gene Ribosomal protein GaRPL18 Salicylic acid
Verticillium dahliae Kleb. is a destructive phytopathogenic fungus that causes wilt diseases on more than 400 plant species, including cotton (Gossypium arboreum) [1, 2]. Verticillium dahliae infects cotton by penetrating the roots. It then spreads across the root cortex and invades the xylem vessels where it forms the conidia responsible for the colonization of vascular tissues and functional impairment. This results in several symptoms, including wilting, discoloration, necrosis, and defoliation [3–6]. Cotton fiber quality and annual yields decrease as a result of Verticillium wilt induced by V. dahliae, and a severe outbreak can lead to yield losses of more than 30% [7, 8]. In China, more than 40% of the cotton-growing area is threatened by Verticillium wilt, potentially causing considerable decreases in cotton production and serious economic losses each year. Furthermore, the fungus can survive for long periods in the soil even without a host, making Verticillium wilt difficult to control using practical and effective chemical treatments [9, 10]. Numerous methods are used to reduce the incidence of Verticillium wilt, such as the application of tillage, soil solarization, soil amendments, and biological controls. However, these are not always efficient or effective [11, 12]. Soil fumigation, which is by far the most effective treatment for inhibiting the propagation of Verticillium species, is costly and can have lethal effects on human health and the environment [7, 13]. The identification and isolation of disease-responsive candidate genes, along with the development of disease-resistant transgenic cotton cultivars, are essential for managing Verticillium wilt [14–16].
The ribosomal protein (RP) has complex structures that differ in prokaryotes and eukaryotes. The eukaryotic ribosome is composed of two unequal subunits (60S and 40S), four ribosomal RNAs (rRNAs), and 82 different RPs. The small ribosomal subunit is composed of a single 18S rRNA and approximately 33 proteins, while the large subunit comprises 28S/25S, 5.8S, and 5S rRNAs, as well as approximately 49 proteins [17–19]. The ribosome is a highly conserved protein that is essential for cellular activities. Although its main function is to synthesize proteins, recent in-depth studies have revealed that it is also important for cell growth, division, and development, and gene regulation [20–22]. Recently, a study has shown that overexpression of the N-terminal 99 amino acids of ribosomal protein L3 confers resistance to pokeweed antiviral protein and the Fusarium mycotoxin deoxynivalenol in tobacco . Another study has shown that ribosomal protein L12 and ribosomal protein L19 are important in nonhost disease resistance in N. benthamiana and A. thaliana. In addition, these genes also play a minor role in basal resistance against virulent pathogens . In particular, a recent study examining ribosomal protein S14 (RPS14) and cancer concluded that this protein can specifically interact with murine double minute 2 (MDM2) to inhibit the degradation of p53 by MDM2 ubiquitin, thereby promoting p53 activity. In gastric and colorectal cancer cells the cell cycle is arrested and tumor cell growth is inhibited in the presence of abundant RPS14 . Another study revealed that ribosomal protein L4 can also regulate the MDM2–p53 loop to regulate p53 activity . Although these studies suggest that RPs are important for disease resistance, they did not include cotton species. Therefore, a more thorough characterization of the function of the cotton RP may be useful for the breeding of Verticillium wilt-resistant cotton varieties.
Under natural conditions, plants frequently encounter diverse potential pathogens. Plants are constantly evolving to cope with these biotic stresses. For example, plants have evolved an immune system that includes constitutive and inducible defense systems that offer protection from potentially dangerous pathogens [27, 28]. Plants also produce several endogenous signaling molecules that help regulate plant defense responses, including jasmonic acid (JA), salicylic acid (SA), and ethylene (ET), all of which are involved in complex signal transduction networks. These biochemical molecules function cooperatively or antagonistically to increase plant resistance to different pathogens [29–32]. Our study revealed that cotton ribosomal protein L18 (GaRPL18) expression levels can be upregulated by accumulated SA, suggesting that RPs can mediate cotton resistance to Verticillium wilt through the SA signaling pathway. While SA is crucial for plant defenses and acquired systemic resistance, it is predominantly involved in the former [33, 34]. Increased SA levels in plant pathogen-challenged tissues and applications of exogenous SA induce the expression of pathogenesis-related (PR) genes, thereby enhancing resistance to invading pathogens [35, 36]. The activation of plant immune responses is also associated with increases in the production of reactive oxygen intermediates and nitric oxide (NO) levels . While the signal transduction networks underlying all plant response mechanisms are complex, crosstalk between the different signaling molecules and networks provides plants with a powerful means of regulating immune responses [38, 39].
In this study, we focused on determining whether GaRPL18 is important for cotton resistance to Verticillium wilt caused by V. dahliae. Our objective was also to identify the signaling pathway associated with cotton defense responses. To verify the expression of GaRPL18, we harvested G. arboreum ‘HuNanChangDeTieZiMian’ samples at different time points after treatments with V. dahliae, methyl jasmonate (MeJA), SA, or ET. We observed that the GaRPL18 expression level increased significantly following V. dahliae and SA treatments. Moreover, we used virus-induced gene silencing (VIGS) technology and transgenic Arabidopsis thaliana lines overexpressing GaRPL18 to functionally characterize GaRPL18 in cotton. Complementary physiology and molecular experiments confirmed that GaRPL18 significantly contributes to cotton resistance against the fungal wilt pathogen V. dahliae via a mechanism related to the SA signaling pathway. Our findings provide insights into the molecular features and functions of a cotton RP gene related to increased resistance to Verticillium wilt.
Plant sources and growth conditions
Seeds of G. arboreum ‘HuNanChangDeTieZiMian’ (resistant) and ‘NaShangQuXiaoHua’ (susceptible) were obtained from the Institute of Cotton Research of the Chinese Academy of Agricultural Sciences. The GaRPL18 overexpression vector (i.e., 35S::GaRPL18) was inserted into wild-type (WT) A. thaliana Columbia ecotype (Col-0) plant. The transgenic plants transformed with 35S::GaRPL18 were grown in a greenhouse at 22 °C and 60% relative humidity under a 16-h light/8-h dark photoperiod. The seeds from different G. arboreum cultivars were incubated in another greenhouse at 25 °C and 80% relative humidity under a 16-h light/8-h dark photoperiod.
Culturing of Verticillium dahliae and inoculation of plants
An antagonistic defoliating Verticillium dahliae isolate (Vd07038) was grown on potato dextrose agar medium at 25 °C for 6 days. Colonies were then cultured in Czapek’s medium [3% (w/v) sucrose, 0.2% (w/v) NaNO3, 0.05% (w/v) MgSO4 · 7H2O, 0.05% (w/v) KCl, 0.002% (w/v) FeSO4 · 7H2O, and 0.131% (w/v) KH2PO4] for 5–7 days at 25 °C with shaking (150 rpm). For V. dahliae treatments, 10 ml conidial suspensions (107 conidia/ml in sterile distilled water) were applied to the bottom of pots containing seedlings. Similarly, A. thaliana plants were grown for 20 days before a 10-ml conidial suspension was injected into the soil using a sterile needle. Control plants were inoculated with an equal volume of sterile distilled water. For in vitro treatments, seedlings were inoculated with V. dahliae, and the extent of stunting was determined using a previously described method . Seedlings were inoculated with a 2-μl conidial suspension (5 × 103 conidia/ml) 2 weeks after germinating.
cDNA cloning and construction of the GaRPL18 overexpression construct
primers used in the research
Forward and reverse primers(5’-3’)
We used the National Center for Biotechnology Information online BLAST tool to analyze the GaRPL18 sequence (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The Gene Structure Display Server 2.0 was then used to analyze gene structures. We also used the ExPASy online tool (http://web.expasy.org/compute_pi/) to predict the isoelectric point and molecular weight. An image of the 3D structure was developed with the PyMOL program (http://www.pymol.org/).
Generation of the virus-induced gene silencing construct and pathogen inoculation
We used Cotton leaf crumple virus (CLCrV)-based vectors (i.e., pCLCrVA and pCLCrVB) for VIGS, with CLCrV:CHLI (encoding magnesium chelatase subunit I) as a positive control [41, 42]. The GaRPL18 fragment was amplified by polymerase chain reaction (PCR) using ‘HuNanChangDeTieZiMian’ cDNA and the VGaRPL18-F/VGaRPL18-R primers (Table 1). The PCR product was digested with SpeI and AscI (BioLabs) and inserted into the pCLCrVA vector. The constructs (i.e., pCLCrVA-GaRPL18, pCLCrVA, and pCLCrVB) were then used to transform A. tumefaciens strain GV3101. The cotyledons of 7-day-old Verticillium wilt-resistant cotton seedlings were then injected with equal amounts of CLCrV vectors. After a 24-h incubation in darkness, the cotton seedlings were transferred to the greenhouse and inoculated with V. dahliae,10 days after the vector infiltration.
Arabidopsis thaliana transformation and molecular analysis
Agrobacterium tumefaciens strain GV3101 containing the GaRPL18 overexpression vector was used to transform Arabidopsis Col-0 via the floral dip method . The T0 transgenic seeds were then spread evenly over soil in a pot. After 1 week, seedlings were sprayed with 0.1% Basta to select positive transformants. The false-positive seedlings turned yellow before dying. Transgenic seeds of the T1 generation were selected on plates of Murashige and Skoog (MS) medium containing 0.1% Basta. After a few days, lines with segregation ratios of approximately 3:1 (i.e., Basta resistant: Basta sensitive) were used to generate T2 lines. The transgenic seeds of the T2 generation were also selected on MS medium containing Basta to identify T3 homozygous lines. The T3 lines with the transgene and the correct segregation ratio were detected based on quantitative reverse transcription (qRT)-PCR analysis of GaRPL18 expression. Only stable homozygous T4 lines exhibiting high GaRPL18 expression levels were chosen for further functional analyses.
Quantitative reverse transcription polymerase chain reaction
We extracted total RNA from the roots and leaves of cotton plants as well as from the leaves of GaRPL18-overexpressing A. thaliana and WT plants using the RNAprep Pure Plant Kit. The RNA was used to prepare cDNA with the PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time; Takara). The Gahistone3 (Cotton_A_11188) and ubiquitin10 (accession: At4g05320) genes were used as internal controls for cotton and A. thaliana, respectively. We designed all qRT-PCR primers with the Primer Premier 6.0 program (Table 1). Diluted cDNA was used as the template for the qRT-PCR, which was conducted with SYBR® Premix Ex Taq™ (Tli RNaseH Plus; Takara) and an ABI 7900 qRT-PCR System (Applied Biosystems, CA, USA). The three-step method involved the following PCR conditions: 40 cycles of 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 30 s. We analyzed the dissociation curves for each reaction and used the 2−ΔΔCT method  to calculate the expression level of each target gene. All reactions were conducted with at least three biological replicates.
Quantification of Verticillium dahliae colonization
We used a previously described qRT-PCR approach to detect and quantify V. dahliae colonization. The qRT-PCR analysis with the V-QPCR-F and V-QPCR-R primer pair (Table 1) was completed as previously described .
Methyl jasmonate, salicylic acid, and ethylene treatments
Plants were treated with 1 mM MeJA, SA, or ET solutions. Cotton or A. thaliana seedlings were grown in pots incubated in a greenhouse. They were sprayed with different solutions at the foliar stage. Control plants were treated with water at the same pH.
Measurements of free salicylic acid, nitric oxide, H2O2, and catalase levels
The abundance of the immune system-related molecules SA, NO, H2O2, and catalase (CAT) was monitored using different methods. The free SA content was determined via the Rigol L3000 high performance liquid chromatography system (Beijing, China) as previously described . We ground leaf samples in liquid nitrogen for subsequent measurements of NO, H2O2, and CAT levels using a Quantitative Assay Kit (Nanjing Jiancheng, Beijing, China).
Cell death assay
Plant cell death was visualized with trypan blue staining as previously described with several modifications . Leaves were soaked in trypan blue dye (1 g phenol, 1 mg trypan blue, 1 ml lactic acid, and 1 ml glycerol dissolved in 1 ml sterile distilled water) and then stained by boiling. After cooling to room temperature, samples were decolorized with a chloral hydrate solution (2.5 g/ml).
Verticillium dahliae recovery assay
To determine the effects of a V. dahliae infection on cotton and A. thaliana plants, we analyzed stem fragments from the first stem node. The cotton and A. thaliana stems were 4.5 cm and 3 cm long, respectively. The stems were cleaned according to a previously described method , and then sliced into six parts. The stem fragments were placed on potato dextrose agar in plates, which were incubated at 25 °C. Plant susceptibility to infection was defined according to the number of stem sections from which the fungus grew.
Analysis of the disease index, stunting, and chlorosis
For cotton plants, the disease index (DI) was calculated according to the following formula: DI = [(∑disease grades × number of infected plants)/(total checked plants × 4)] × 100%. Seedlings were classified into five grades (i.e., grade 0, 1, 2, 3, and 4) according to the symptoms on the cotyledons and true leaves [49, 50]. The disease severity for A. thaliana plants was graded on a 0–5 scale, and the DI was calculated with the following formula as previously described : DI = [(∑disease grades × number of infected plants)/(total checked plants × 5)] × 100%. The DI represents a comprehensive and objective measure of plant health, with high DI values corresponding to serious infections. The extent of stunting was rated on a 0–3 scale (0 = no stunting; 1 = moderate reduction in leaf area; 2 = considerable decrease in leaf area; and 3 = considerable decrease in leaf area, leaf number, and stem length). Leaf chlorosis was rated on a 0–4 scale (0 = no symptoms; 1 = up to 25% chlorotic leaves; 2 = up to 50% chlorotic leaves; 3 = up to 75% chlorotic leaves; and 4 = up to 100% chlorotic leaves) .
Analysis of GaRPL18 structure and expression patterns in cotton plants treated with Verticillium dahliae or hormones
Interactions between GaRPL18 and PR genes
Silencing of GaRPL18 in cotton considerably weakened resistance to Verticillium dahliae
Salicylic acid, nitric oxide, H2O2, and catalase levels decreased in GaRPL18-silenced cotton plants upon Verticillium dahliae infection
To further evaluate the role of GaRPL18 and its relationship with the SA pathway in cotton defense responses to V. dahliae infection, we analyzed the SA content and the abundance of several other immune-responsive compounds (i.e., NO, H2O2, and CAT) in V. dahliae-treated plants. All compounds were present in lower amounts in inoculated CLCrV:GaRPL18 plants than in CLCrV control plants (Fig. 4c, d). These results indicate that silencing GaRPL18 in wilt-resistant cotton plants inhibits V. dahliae-induced production of SA, NO, H2O2, and CAT, further confirming that GaRPL18 is closely related to the SA signaling pathway.
Transgenic Arabidopsis thaliana seeds overexpressing GaRPL18 increased in size and weight, while seedlings became more resistant to Verticillium dahliae
Effects of GaRPL18 overexpression on the salicylic acid signaling pathway in Verticillium dahliae-treated transgenic Arabidopsis thaliana plants
We examined the expression level of signaling pathway-related genes, including those involved in JA biosynthesis [i.e., octadecanoid-responsive Arabidopsis AP2/ERF domain protein 59 (AtORA59), plant defensin 1.2 (AtPDF1.2), and vegetative storage protein 2 (AtVSP2)], SA biosynthesis [i.e., non-expressor of pathogenesis-related genes 1 (AtNPR1), pathogenesis-related gene 1 (AtPR1), and pathogenesis-related gene 3 (AtPR3)], and ET biosynthesis [i.e., amino cyclopropane carboxylate oxidase 2 (AtACO2), ethylene insensitive 3 (AtEIN3), and ethylene response factor 1 (AtERF1)]. As shown in Fig. 5D, over-expression of GaRPL18 in the Col-0 background did not affect expression of genes related to the JA, SA and ET pathways; these marker genes had similar expression levels in WT and over-expression plants without V. dahliae stress. In addition, the expression levels of the JA- and ET-related marker genes were similar overall between the WT controls and GaRPL18- overexpressing transgenic A. thaliana plants at 24 h post-inoculation (hpi). However, the SA-related genes were expressed more highly in the rosette leaves of transgenic plants than in the WT leaves at 24 h post-inoculation. The expression levels in the WT leaves were unchanged (Fig. 5d). Thus, GaRPL18 overexpression had little effect on the signal transduction pathways regulating JA and ET biosynthesis, but had a significant effect on the SA biosynthesis signal transduction pathway.
Application of exogenous salicylic acid increases the resistance of transgenic Arabidopsis thaliana plants to Verticillium dahliae
We calculated the DI values and quantified the V. dahliae colonization using qRT-PCR for WT plants, SA-treated WT plants, transgenic plants, and SA-treated transgenic plants. The DI value for WT plants was significantly higher than that of transgenic plants, including those treated with SA (Fig. 6d). Analyses of the expression level of fungal genes revealed that SA-treated transgenic plants were the most disease-resistant, while WT plants were the most susceptible (Fig. 6e). The trypan blue staining results were similar for WT and SA-treated WT plants. We observed dark blue veins and large stained mesophyll areas. The veins of transgenic leaves were light blue, and the mesophyll was not extensively stained. These observations are consistent with the greater disease resistance of SA-treated transgenic plants (Fig. 6f).
Verticillium wilt is one of the most lethal fungal diseases of plants and significantly reduces the quality and annual yields of cotton. Therefore, clarifying the molecular mechanism underlying cotton resistance to V. dahliae is essential for developing new wilt-resistant cotton varieties. Previous studies involving V. dahliae infections revealed that this pathogen attacks susceptible plants by germinating on the roots. It then employs various mechanisms to move through the root cortex into the plant vascular system, allowing for further colonization of the plant. When the fungus has completely infected the plant, it produces microsclerotial spores that remain stable and dormant in the soil until they are exposed to a new host . To breed new cotton varieties resistant to Verticillium wilt, more research is required to identify resistance-related genes. Technological improvements have facilitated the mining and functional analysis of genes. There are some reports that describe the application of genes encoding proteins associated with resistance to V. dahliae to develop transgenic A. thaliana plants capable of inhibiting disease development. These genes include those encoding glutathione S-transferase [54, 55], rate-limiting enzymes , and transcriptional regulators . However, efforts involving gene mining have been insufficient, and the cotton-producing regions of China are still affected by annual outbreaks of Verticillium wilt. The lack of known wilt-resistance genes in cotton makes the identification and functional characterization of V. dahliae resistance-related genes essential for the development of new wilt-resistant cotton varieties. In this study, we confirmed that GaRPL18, which encodes an RP, influences resistance to Verticillium wilt.
The function and characteristics of RPs during protein synthesis have been thoroughly researched, and recent in-depth studies confirmed that RPs also have important roles in other processes [20–22]. In particular, some studies have shown PRs play an important role in plant disease resistance [23, 24]. Recent cancer studies concluded that the RPs can regulate the expression of key genes in the MDM2–p53 regulatory loop, thereby promoting p53 activity and suppressing tumor growth [25, 26, 58–60]. Additionally, the RP has other effects related to disease resistance. For example, mutations to RPS19 are associated with a congenital erythroblastopenia with a decreased abundance or lack of erythroid precursors . However, little is known about the function and characteristics of RP-encoding genes during cotton resistance to V. dahliae. In this study, we determined that the RP mediates the resistance of cotton plants to V. dahliae.
To confirm the role of GaRPL18 in cotton pathogen defense responses, we cloned the gene using cDNA produced from RNA extracted from wilt-resistant cotton plants. We also analyzed the GaRPL18 expression patterns in different cotton varieties treated with V. dahliae. The GaRPL18 expression level was rapidly and considerably upregulated in infected wilt-resistant cotton plants, but was relatively unchanged in the wilt-susceptible cotton plants. We then examined the expression of PR genes in wilt-resistant cotton plants. We confirmed that cotton resistance to V. dahliae results from interactions among various disease resistance systems that function together to resist pathogen invasions. Using VIGS technology, we determined that the silencing of GaRPL18 in wilt-resistant cotton plants significantly decreased resistance to V. dahliae. Additionally, the NO, H2O2, and CAT contents were lower in cotton plants in which GaRPL18 was silenced than in vector-control plants. An invasion by V. dahliae induces a spike in NO content, which is important for disease resistance in plants . An earlier study concluded that the H2O2 content is significantly higher in disease-resistant tomato plants than in control plants, and that CAT plays an important role in early defense responses . Our results indicate that NO, H2O2, and CAT affect the GaRPL18- related responses to V. dahliae in cotton plants. These observations confirm that GaRPL18 is important for cotton resistance to V. dahliae, which is consistent with the anti-carcinogenic effects reported for other RPs. We used transgenic A. thaliana plants to further analyze V. dahliae disease resistance traits based on DI values, V. dahliae colonization, trypan blue staining, and recovery assays. Moreover, while the rosette leaves of WT plants gradually exhibited disease symptoms with increasing fungal growth and spread, almost no disease symptoms were observed on the rosette leaves of GaRPL18-overexpressing transgenic plants. This implies that the fungus was inhibited in the roots of the transgenic plants, and prevented from spreading into the rosette leaves. Additionally, in vitro analyses of the wilt-resistance of GaRPL18-overexpressing A. thaliana plants further confirmed the role of GaRPL18 in plant defense responses to V. dahliae. This suggests that the accumulation of GaRPL18 considerably impedes pathogen colonization, even at the seedling stage. In summary, we confirmed that GaRPL18-overexpressing transgenic plants were more resistant to V. dahliae than WT plants, and that the extent of V. dahliae colonization was significantly lower in transgenic plants than in WT plants.
We also observed that GaRPL18 expression was rapidly upregulated by SA, reaching peak levels 6 h after the application of the hormone. In contrast, MeJA and ET treatments had minimal effects on GaRPL18 expression. Therefore, we hypothesized that GaRPL18 may be associated with the SA signaling pathway. This was verified by a significant decrease in SA levels in V. dahliae-infected CLCrV:GaRPL18 plants. The activation of SA signaling in stressed plants can stimulate the expression of downstream disease-resistance genes to provide protection from pathogens [64, 65]. There are many genes related to the SA signaling pathway, including NPR1, PR1, PR3, WRKYs, and AtGSTF6 [66–69]. To verify these results, the JA-, SA- and ET-related marker gene expression levels were examined in V. dahliae-infected transgenic A. thaliana plants overexpressing GaRPL18. The upregulated expression of SA signaling pathway-related genes (i.e., AtNPR1, AtPR1, and AtPR3) and SA content confirmed our hypothesis. To further clarify the link between GaRPL18 and SA signaling, we treated the transgenic and WT plants with exogenous SA, and observed that the hormone significantly increased the resistance of transgenic plants to V. dahliae. These findings imply that GaRPL18, working in concert with the SA signaling pathway, has a strongly antimicrobial effect on V. dahliae. Therefore, GaRPL18 may be useful for breeding Verticillium wilt-resistant cotton varieties.
To the best of our knowledge, this study is the first to examine the RP function related to cotton resistance to V. dahliae. We used VIGS technology to confirm that GaRPL18 is important for the resistance of cotton plants to V. dahliae infections. Our data also suggest that breeding new cultivars that overexpress GaRPL18 may be an effective way to control Verticillium wilt of cotton plants. Finally, this study revealed that SA is an important factor related to the cotton defense response system, and that the mechanism of GaRPL18-associated V. dahliae resistance is related to the SA signaling pathway.
4-coumarate: CoA ligase
Amino cyclopropane carboxylate oxidase 2
Cotton leaf crumple virus
Ethylene insensitive 3
Ethylene response factor 1
Murine double minute 2
Nonexpressor of pathogenesis-related gene 1
Octadecanoid-responsive Arabidopsis AP2/ERF domain protein 59
Phenylalanine ammonia lyase
Plant defensin 1.2
Virus-induced gene silencing
Vegetative storage protein 2
We thank the members of Professor Zhu’s groups (Institute of Cotton Research, Chinese Academy of Agricultural Sciences) for providing the V. dahliae isolate and their technical assistance.
This work was supported by The Major Program of Joint Funds (Sinkiang) of the National Natural Science Foundation of China (Grant No. U1303282) and the Zhengzhou Science and Technology Program (Grant No. 153PXXCY180). The funding bodies were not involved in designing the study, the collection, analysis, and interpretation of data, or the preparation of the manuscript.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files.
Conceived and designed the experiments: QG, ZY, SH, and FL. Conducted the experiments: QG and ZY. Analyzed the data: QG and ZY. Contributed reagents/materials/analysis tools: QW, HIB, JZ, YZ, and EC. Wrote the manuscript: QG. All authors have read and approved the current version of the manuscript.
The authors declare that there are no competing interests.
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