Isolation and characterization of the GbVIP1 gene and response to Verticillium wilt in cotton and tobacco

Abstract

Background

Verticillium wilt is a serious soil-borne vascular disease that causes major losses to upland cotton (Gossypium hirutum L.) worldwidely every year. The protein VIP1 (VirE2 interaction protein 1), a bZIP transcription factor, is involved in plant response to many stress conditions, especially pathogenic bacteria. However, its roles in cotton response to Verticillium wilt are poorly understood.

Results

The GbVIP1 gene was cloned from resistant sea-island cotton (G. barbadense) cv. Hai 7124. Expression of GbVIP1 was up-regulated by inoculation with Verticillium dahliae and exogenous treatment with ethylene. Results of virus-induced gene silencing suggested that silencing of GbVIP1 weakened cotton resistance to Verticillium wilt. The heterologous expression of GbVIP1 in tobacco showed enhanced resistance to Verticillium wilt. The PR1, PR1-like and HSP70 genes were up-regulated in GbVIP1 transgenic tobacco after Verticillium wilt infection.

Conclusion

Our results suggested that GbVIP1 increased plant resistance to Verticillium wilt through up-regulating expressions of PR1, PR1-like, and HSP70. These results provide new approaches to improving resistance to Verticillium wilt in upland cotton and also have great potential for disease-resistance breeding of cotton.

Background

Over 200 kinds of dicotyledonous plant species are susceptible to Verticillium wilt, a serious soil-borne vascular disease (Fradin and Thomma 2006). Among them, upland cotton (Gossypium hirsutum L) is the most economically important crop for natural textile fiber and oil in the world. Cotton infected with Verticillium wilt shows several symptoms including leaf wilting or defoliation and vascular discoloration or necrosis. Verticillium wilt causes enormous losses in cotton yield and fiber quality every year in many couZntries, especially China (Cai et al. 2009). There are no effective chemical and biological methods to control the contagion of Verticillium wilt because of its highly aggressive pathogenicity and many years of survival in soil as resting structures without a host (Gong et al. 2018). After expending great efforts on control of Verticillium wilt, it was suggested that cultivating resistant varieties was an economical and effective method of control. Unfortunately, upland cotton, the most widely cultivated cotton species, is usually susceptible to Verticillium wilt (Zhang et al. 2012b). The related sea-island cotton (G. barbadense) is often highly resistant or tolerant to Verticillium wilt (Zhou et al. 2014). Nevertheless, G. barbadense cannot be vigorously promoted for cultivation due to yield limitations. Due to difficulty in performing interspecific crosses of G.hirsutum and G. barbadense and the low efficiency of traditional breeding, development of Verticillium disease-resistant cultivar breeding has been slow (Zhang et al. 2014). Therefore, many researchers have focused on the key genes involved in the disease-resistance process and bred disease-resistant cotton cultivars via modern genetic engineering and molecular breeding methods. Genes associated with Verticillium wilt resistance were identified using map-based cloning, genome-wide association study and high-throughput sequencing, including NBS-LRR genes (e.g. GbaNA1 and GbRVd) (Li et al. 2018; Yang et al. 2016), kinase and receptor-like protein genes (e.g. GhMKK2 and GbRLK) (Jun et al. 2015) and synthesis genes of antitoxin and antifungal protein (e.g. GhPAO and GhAFP4) (Mo et al. 2015; Wang et al. 2016). The resistance process to Verticillium wilt in cotton is very complex and a series of genes and proteins are regulated to from the defense response. As the cotton genome was sequenced, increasing numbers of genes involved in the process of resistance to Verticillium wilt were identified and cloned (Zhao et al. 2018). However, it is important to find the major resistance gene. Transcription factors associated with disease resistance, which regulate expression of resistance genes might play a key role in plant defense against pathogens.

The protein VIP1 (VirE2 interacting protein1), was first identified in Arabidopsis as a host protein that specifically interacted with the VirE2 protein of Agrobacterium tumefaciens (Tzfira et al. 2000). VIP1 is involved in the formation of T-DNA complexes and assists T-DNA in cytoplasm transporting, nuclear importing and nuclear locating (Liu et al. 2010). The VIP1 is a plant transcription factor belonging to subfamily I of the bZIP (basic-zipper protein) transcription factor family and contains a leucine zipper domain (Jakoby et al. 2002). A nuclear locating signal is located in the leucine zipper domain of VIP1 (van der Krol and Chua 1991). As a transcription factor, VIP1 regulates a series of gene expressions via combining the VIP1 response element region and response to biotic and abiotic stresses including pathogenic bacteria, drought, touch, low-sulfur and hyperosmotic stress (Lacroix and Citovsky 2013). Zaltsman et al. (2010) suggested that VIP1 was a defense-related transcription factor which up-regulated the expression of PR1 (pathogenesis-related protein 1). When the plant suffered from pathogenic bacteria, Ser⁷⁹ of VIP1 was phosphorylated by mitogen-activated protein kinase in the cell cytoplasm. Phosphorylated VIP1 then translocated to the cell nucleus and activated PR1 transcription, which finished the plant disease-resistance response. Tsugama et al. (2014) found that AtVIP1 accumulated in the nucleus during hypo-osmotic stress and confirmed the VIP1-binding sequences (AGCTGT/G) by gel shift assays. The VIP1 regulated the expression pattern of hypo-osmolarity-responsive genes CYP707A1 and CYP707A3, which are involved in the plant response to hypo-osmotic conditions. The VIP1 interacted with heterotrimeric G proteins β subunit (AGB1) during hypotonic conditions but not in mannitol-containing hypertonic conditions, implicating VIP1 and AGB1 in response of Arabidopsis thaliana to osmolarity and/or turgor pressure (Tsugama et al. 2013). Additionally, AtVIP1 acted as a downstream gene of AGB1 and AtVIP1 overexpression in Arabidopsis increased its sensitivity to abscisic acid treatment and enhanced its drought tolerance (Xu et al. 2015). Researchers used a specific transgenic plant in which the genes up-regulated by VIP1 were repressed, to examine the AtVIP1 physiological roles and suggested that AtVIP1 suppressed the root waving induced by touch stress and the phenomenon was influenced by ethylene and auxin (Tsugama et al. 2016a, b). Li et al. (2014) cloned the Agvip1 gene from three celery cultivars and found that Agvip1 was expressed differently in different organs and cultivars. Results suggested that Agvip1 was up-regulated under cold, drought, hot, salt and metal ion stresses in different cultivars. Wu et al. (2010) isolated a low-sulfur-tolerant mutant with a recessive locus in AtVIP1 which improved sulfur utilization efficiency and understanding of the underlying molecular mechanism of sulfur tolerance using VIP1.

It is clear that VIP1, as one kind of bZIP transcription factor, is involved in many responses to stress, especially of pathogenic bacteria. Thus, we planned to utilize VIP1 to increase the resistance of upland cotton to Verticillium wilt. In this study, GbVIP1 which encodes a bZIP transcription factor protein was cloned in G. barbadense. The structure, expression pattern and hereditary character of GbVIP1 were investigated. The primary biological function of GhVIP1 was ascertained in cotton and tobacco using VIGS and transformation strategy. Our output will provide new approaches to improve resistance to Verticillium wilt in upland cotton and will also have much potential in disease-resistance breeding of cotton.

Results

GbVIP1 cloning and structure analysis

The AtVIP1 protein sequence (Q9MA75) was applied as a seed sequence to BLAST NCBI databases with the organism selected as cotton. A predicted transcription factor VIP1-like gene (XM016838286) was identified. According to sequence information, we designed primers to amplify the VIP1 from G. barbadense cv. Hai 7124. A 1 014 bp fragment was obtained and sequence analysis indicated that GbVIP1 encoded a polypeptide of 337 amino acid residues. The protein sequence of GbVIP1 had 49% similarity with AtVIP1. Mapping the 1 014 bp cDNA sequence identified two copies of GbVIP1 in the G. barbadense genome database. One was mapped to chromosome D02 and localized to the reference genome within positions 10 550 650–10 553 567. The other copy was mapped to chromosome A02 and localized within positions 10 056 074–10 062 958. The alignment results showed that GbVIP1 contained four exons and three introns. To determine the sequence differences among cultivars resistant and susceptible to Verticillium wilt, VIP1 was also amplified from G. hirsutum cv. TM-1 and G. barbadense cv. Pima. We found eight amino acid differences among VIP1 protein sequences from cv. TM-1 and Hai 7124 (Fig. 1a). The VIP1 protein sequences of other plant species were downloaded from NCBI to investigate the phylogenetic relationships and conservatism of the bZIP domain among different species. The phylogenetic tree showed VIP1 from tetraploid and diploid cotton clustered into a subgroup, and VIP1 from Glycine max and Citrus sinensis had a close phylogenetic relationship with VIP1 from cotton (Additional file 1). The conserved domain of VIP1 from different plant species was predicted by CD-Search software and the results suggested that all examples of the VIP1 protein had one bZIP domain (Fig. 1b). We extracted the protein sequences of the VIP1 conserved domain and analyzed the conservation of amino acids using WebLogo software. The bZIP domain from different plant species was had highly conserved and there was one leucine every six amino acids (Fig. 1c).

Fig. 1

The VIP1 protein sequences from three cotton cultivars and analysis of bZIP domain in VIP proteins. a Amino acid differences among VIP1 protein sequences from cvs TM-1, Hai 7124 and Pima. b structural analysis of VIP1 proteins from different plant species. c Sequence logos showing the highly conserved bZIP domain: one leucine every six amino acids

Expression patterns of VIP1 gene

Quantitative real-time PCR (qRT-PCR) was used to determine the expression profile of VIP1 in different tissues (roots, stems and leaves) of TM-1 and Hai 7124. Results indicated that VIP1 was expressed in all of the tested tissues but was highly expressed in roots and little expressed in stems. Tissue specific expression results from the two cultivars were consistent (Fig. 2a). To investigate the expression pattern of VIP1induced by Verticillium dahliae, the relative expression level of VIP1 was determined after inoculation with V. dahliae. Expression of VIP1 was up-regulated at 6 h after inoculation. At 24 h after inoculation, VIP1 expression began to return to its original level (Fig. 2b). Using qRT-PCR to evaluate the effect of plant hormone (salicylic acid (SA, Ethylene (ET) and Jasmonic acid (MeJA) treatments showed that VIP1 expression was up-regulated under exogenous treatment of ET but the other hormones had no significant effect on expression (Fig. 2c).

Fig. 2

Expression patterns of VIP1 gene under different conditions. a Tissue-specific expression of VIP1. b Expression pattern of VIP1 induced by V. dahliae. c Expression pattern of VIP1 under exogenous treatment of plant hormones

Silencing of GbVIP1 reduced Verticillium wilt resistance in cotton

A tobacco rattle virus (TRV)-based VIGS system was used to study the function of GbVIP1 in cotton responses to Verticillium wilt. We designed the appropriate primer and amplified a GbVIP1 fragment of about 300 bp. The selected fragment was integrated into pTRV2 vector to generate VIP1-knockdown cotton lines. After Agrobacterium infection, silencing efficiency was assessed using qRT-PCR. The relative expression level of VIP1 was reduced severely in TRV: VIP1 compared with the control, and silencing efficiency exceeded 70% (Fig. 3a). When TRV:PDS seedlings showed an albino phenotype for new leaves, all the silenced cotton seedlings were inoculated with V. dahliae (Fig. 3b). About 15 days after inoculation, symptoms of Verticillium wilt were observed in TRV:VIP1 seedlings, including yellow, wilting and falling leaves, whereas the control showed no symptoms (Fig. 3b). We investigated the disease index (DI) values of TRV:VIP1 and control seedlings at 25 and 35 days after inoculation. The DI values of TRV:GbVIP1 and TRV:00 did not significantly differ at 25 days after inoculation. However, the DI value of TRV:VIP1 seedlings was about 30 at 35 days after inoculation, which was remarkably higher than 23.45 for control (Fig. 3c). Leaves of silenced and control cotton seedlings were picked off and showed more disease speckles from silenced seedlings than control (Fig. 4a). We extracted the stems of silenced and control seedlings and cut through the middle of the stem vascular bundle. The xylem of TRV:VIP1 seedlings showed more brown and necrotic areas than control because V. dahliae infected the plants through vascular bundles (Fig. 4b). These stems were also used in recovery experiments to analyze the level of V. dahliae colonization. Results indicated that more fungal growth around stems from TRV:VIP1 than control seedlings and suggested that TRV:VIP1 seedlings suffered more severe disease (Fig. 4c). Leaves of silenced cotton seedlings were dipped in trypan blue dye to assay the cell state of plants after V. dahliae inoculation. The leaves of TRV:VIP1 seedlings had larger and darker blue areas, gathered around veins than controls (Fig. 4d), indicating that V. dahliae inoculation caused more dead cells in leaves of TRV:VIP1 seedlings. The above results suggest that VIP1 silencing weakened cotton resistance to Verticillium wilt.

Fig. 3

Silencing GbVIP1 gene resulted in decreased Verticillium wilt resistance in cotton. a Expression levels of GbVIP1 in silenced and wild type cotton seedlings. b Phenotypes of cotton seedlings after V. dahliae inoculation. TRV:00 represents empty vector, TRV:GbVIP1 represents GbVIP1 silenced and TRV:PDS represents PDS silenced. c Disease index of silenced and wild type cotton seedlings measured at 25 and 35 days after infection

Fig. 4

Leaf and stem sections from GbVIP1 silenced and control cotton seedlings. a Detached leaves from GbVIP1 silenced and control cotton seedlings after V. dahliae infection. b Stem sections from GbVIP1 silenced and control cotton seedlings after V. dahliae infection. c Recovery experiments of stem sections from GbVIP1 silenced and control cotton seedlings after infection of V. dahliae infection. d Trypan blue dye experiments of leaves from silenced and control cotton seedlings after infection of V. dahliae infection

Overexpression of GbVIP1 increased resistance to Verticillium wilt in tobacco

To further study the function of VIP1, tobacco cv. NC89 was used to overexpress cotton VIP1. The VIP1 was inserted into the pBI121 vector driven by 35S promoter and transformed into tobacco by Agrobacterium-mediated transformation. Then PCR and qRT-PCR were used to verify transgenic plants and the results showed that GbVIP1 was significantly up-regulated in transgenic tobacco plants (Additional file 2). Two approaches were used to determine the disease-resistance capacity of transgenic tobacco. Detached leaves from transgenic and control tobacco plants were inoculated with V. dahliae and, 16 days after inoculation, leaves from control plants showed obvious disease symptoms compared with transgenic plants (Fig. 5a). The transgenic tobacco plants had a lower disease grade of detached leaves than control (Fig. 5b). Additionally, following treatment with V. dahliae, transgenic tobacco showed more resistance to Verticillium wilt than control seedlings (Fig. 5c and d). Hence, GbVIP1 from cotton conferred resistance to Verticillium wilt in tobacco plants. To find the possible mechanism underlying VIP1 increasing the disease resistance in cotton, qRT-PCR was used to determine expression levels of several possible resistance-related genes in transgenic and control tobacco: PR1, PR1-like, RAR1, HSP70 and RPP13. All selected resistance-related genes showed increased expression at 6 h after inoculation and PR1 and PR1-like were significantly up-regulated in GbVIP1-overexpressing tobacco seedlings (Fig. 6). The transcript abundances of PR1 and PR1-like increased over 10-fold in transgenic tobacco after inoculation with V. dahliae compared with control, suggesting that GbVIP1 overexpression activated expression of defense genes.

Fig. 5

GbVIP1 gene from cotton conferred resistance to Verticillium wilt in transgenic tobacco plants. a Disease phenotype of leaves from GbVIP1 transgenic tobacco and control at 16 days after inoculation with V. dahliae. 35S:GbVIP1 represents transgenic tobacco lines and CK represents the control tobacco lines. b Extent of chlorosis of leaves. c Whole plant disease phenotype of GbVIP1 transgenic tobacco and control at 25 days after infection of V. dahliae. d Disease index of GbVIP1 transgenic tobacco and control seedlings measured at 25 and 35 days after infection

Fig. 6

Expression level of disease resistance-related genes in transgenic tobacco plants expressing GbVIP1. 35S:GbVIP1 represents transgenic tobacco lines and CK represents control tobacco lines. * Significant differences

Discussion

Verticillium wilt has become the most serious and devastating disease of cotton in China and causes heavy losses of cotton production. Many years of practicing disease prevention has shown that breeding and application of disease-resistant cotton cultivars are the most effective and economical methods to reduce damage from Verticillium wilt to cotton. However, upland cotton cultivars with high resistance to Verticillium wilt are rare. The common cultivated cotton varieties are allotetraploid and the cotton genome is huge, both of which limit development of Verticillium disease-resistant cultivars. Screening of resistant genes and their utilization using modern molecular biology has accelerated the breeding of disease-resistant cultivars. The function and underlying mechanisms of VIP1 protein in disease and stimuli responses have been researched in model plants such as Arabidopsis thaliana, but the function of VIP1 in responding to Verticillium wilt is little understood. In this study, we found new roles of GbVIP1 in defense responses to Verticillium wilt and studied the mechanisms underlying the functions of GbVIP1 in cotton.

The GbVIP1 gene was cloned from a resistant G. barbadense variety using the homologous cloning method. The GhVIP1 was also cloned from susceptible G.hirsutum and base differences were found among VIP1 nucleotide sequences from resistant and susceptible varieties, which we speculated resulting in the different disease resistance of varieties. In our research, GbVIP1 was obviously up-regulated by Verticillium wilt inoculation and exogenous treatment of ET in cotton. The process of plant response to pathogenic bacteria is regulated by multiple signals, in which plant hormones play a key role (Katagiri and Tsuda 2010). Previous studies suggested that ET had a multiple effect on the interaction between plants and Verticillium wilt, with several genes activated by ET in response to Verticillium wilt, including ethylene responsive factor (ERF6), ERF1 and GbERF1-like (Robison et al. 2001; Yang et al. 2015; Guo et al. 2016). These transcription factors regulated the expression of downstream resistance proteins and increased the plant disease resistance. We speculated that GbVIP1 was regulated by ET, which played a key role in plant tolerance to Verticillium wilt.

We studied the resistance function of VIP1 through positive and negative sides: the positive was GbVIP1 overexpression in tobacco using Agrobacterium mediated transformation and the negative was silencing of GbVIP1in cotton using VIGS. Results consistently indicated that GbVIP1 overexpression in tobacco increased resistance to Verticillium wilt, but silencing GbVIP1 in cotton decreased the resistance. Thus, GbVIP1 had a positive role in Verticillium wilt resistance.

As described previously, VIP protein is a bZIP transcription factor and regulates expression of a series of stress-related genes. Notably, VIP1 up-regulated PR1 expression in plants infected by pathogenic bacteria. We consistently found that PR1 was obviously up-regulated in transgenic GhVIP1 tobacco at 6 h after incubation of Verticillium wilt. The PR protein family was ubiquitous in plant and play roles in multiple growth and development processes (Kaur et al. 2017). Among these, PR1 is a marker gene of disease-resistance response, and PR1 was also up-regulated by some disease-resistance genes during incubation of Verticillium wilt in cotton (Lu et al. 2011). For instance, Arabidopsis plants overexpressing GhSNAP33 and Gbvdr6 showed resistance to V. dahliae with elevated expression of PR1, and endogenous cAMP induced rapid increases of PR1 transcription in plant defense responses against pathogen Verticillium (Yang et al. 2017; Jiang et al. 2005; Wang et al. 2018). It is possible that GbVIP1 might increase resistance to Verticillium wilt in transgenic tobacco by up-regulating expression of PR1. In addition to PR1, RAR1, HSP70 and RPP13 were also up-regulated during incubation of Verticillium wilt at different levels and different time points. The expression pattern of HSP70 was similar to that of PR1 was significantly up-regulated in GbVIP1 transgenic tobacco at 6 h after infection and expression level was reduced after 12 h. The HSP70 proteins are evolutionarily conserved molecular chaperones and play a key role in correct protein folding, plant growth and development process and biotic and abiotic stress responses in plants (Lin et al. 2001). HSP70 is rapidly up-regulated and accumulates in plants experiencing stress and HSP70 can reduce the damage to plant cells, which improves plant stress tolerance (Sung 2001). In our studies, the GbVIP1 transgenic tobacco may have increased the resistance to Verticillium wilt through up-regulating HSP70. The expression levels of RAR1-like and RPP13 were slightly up-regulated after 6 h infection and reached maxima after 12 h in transgenic tobacco. The RAR1 protein is a eukaryotic zinc-binding protein and RPP13 contains CC, NB-ARC and LRR domains, which play a crucial part in resistance to various plant diseases(Cheng et al. 2018; Wang et al. 2017). In our results, RAR1-like and RPP13 were not significantly up-regulated by GbVIP1 and we deduced that RAR1-like and RPP13 proteins might have little relationship with GbVIP1 and Verticillium wilt.

Conclusions

We cloned GbVIP1 from a resistant G. barbadense variety and verified the resistance function of GbVIP1 in plant defense against Verticillium wilt. Our results suggested that GbVIP1 increased plant resistance to Verticillium wilt through up-regulating the expression levels of PR1, PR1-like and HSP70. Our output will provide new approaches to improve disease resistance to Verticillium wilt in G. hirsutum and also have much potential for disease-resistance breeding of cotton.

Methods

Plant materials and growth conditions

The cotton cv. Hai 7124 (resistant), Pima (resistant) and TM-1(susceptible) were obtained from the Institute of Cotton Research of Chinese Academy of Agricultural Sciences. The tobacco line NC89 was kindly granted by Dr. Pei Xinwu from the Biotechnology Research Institute of Chinese Academy of Agricultural Sciences. Cotton seedlings were grown in incubators at 25 °C during the day and 20 °C at night, 60% relative humidity, under a 16/8 h light/dark photoperiod. The tobacco aseptic seedlings were grown in another incubator at 25 °C and 60% relative humidity conditions under a 16/8 h light/dark photoperiod in culture bottles. Transgenic tobacco seedlings were grown in a greenhouse under the same conditions as cotton seedlings.

V. dahliae materials and inoculation methods

The V. dahliae (Vd853) was kindly provided by Professor Zhu Heqin of the Institute of Cotton Research of Chinese Academy of Agricultural Sciences. The Vd853 was cultured on potato dextrose broth at 25 °C for 6 days with shaking. Then, conidia were harvested and grown in liquid Czapek’s medium at 25 °C for 7 days with shaking. Czapek’s medium comprised 3% sucrose, 0.2% NaNO₃, 0.131% KH₂PO₄, 0.05% KCl, 0.05% MgSO₄·7H₂O and 0.002% FeSO₄·7H₂O (all w/v). The conidia concentration was verified by counting conidia using a hemocytometer under a microscope. For V. dahliae infection, the conidia working concentration was 1 × 10¹⁰ conidia·L^-1. Roots of the cotton or tobacco seedlings were uprooted gently, dipped in 10 mL of conidial suspensions for 5 min and replanted in pots. The V. dahliae infection of tobacco leaves was performed as described previously (Munis et al. 2010). Leaves from transgenic and wild type tobacco plants were harvested at the same position. The detached leaves were inoculated with conidial suspensions (1 × 10¹⁰ conidia·L^-1) for 5 s and then put in sterile Petri dishes with moistened sterile filter paper at 25 °C for 48 h. After incubation, infected leaves were washed three times with sterile demineralized water and then placed in new sterile Petri dishes with moistened sterile filter paper at 25 °C.

Extraction of RNA and gene cloning

Plant total RNA was extracted using EASYspin Plus Plant RNA kit (Aidlab, Beijing, China) according to the manufacturer’s instructions. The cDNA was synthesized using a PrimeScript™ II 1st strand cDNA Synthesis Kit (TakaRa, Dalian, China). For cloning GbVIP1, a 20 μL reaction system was used, containing 10 × PCR buffer for KOD-Plus-Neo, 0.2 mmol·L^-1 dNTPs, 1.5 mmol·L^-1 MgSO₄, 0.3 μmol·L^-1 forward primer and 0.3 μmol·L^-1 reverse primer, 0.4 U of KOD-Plus-Neo (TOYOBO, Osaka, Japan) and 200 mmol·L^-1 cDNA. The PCR amplification was performed on a Bio-Rad PCR thermal cycler (C1000) and the procedure consisted of 94 °C for 4 min, 34 cycles of 98 °C for 10 s, 60 °C for 1 min, 68 °C for 30 s/kb, and 68 °C for 5 min. The PCR product was segregated by agarose gel electrophoresis and purified by TIANgel Maxi Purification Kit (TIANGEN, Beijing, China). Purified product was cloned into the pEASY-Blunt Zero Cloning vector (TransGen, Beijing, China) and sequenced by GENEWIZ (Suzhou, China). The GhVIP1 from upland cotton was cloned and sequenced using the same method. All primers used in this paper are listed in Additional file 3 and were synthesized by GENEWIZ.

Vector construction for virus-induced gene silencing (VIGS) in cotton and VIGS experiments

The GbVIP1 fragment (272 bp) was amplified by VIP1-V-F/ VIP1-V-R primers as described in previous steps. The GbVIP1 fragment was inserted into pYL156 vector, a TRV based vector used for VIGS through ClonExpress™ II One Step Cloning Kit (Vazyme, Nanjing, China). The pYL156–GhPDS used as a positive control vector was constructed using the same method. The plasmids containing pYL156–GbVIP1, pYL156–GhPDS, pYL156 and pYL192 were transformed into Agrobacterium tumefaciens strain GV3101 respectively using the freeze–thaw method (Dupadahalli 2007). For VIGS, Agrobacterium was harvested and injected into two fully expanded cotyledons of cotton seedlings as previously described (Gao and Shan 2013). The VIGS experiments were performed with at least three biological repeats and for each repeat there were more than ten plants per constructed vector.

Vector construction for overexpression in tobacco and Agrobacterium-mediated transformation

The full-length GbVIP1 coding sequence was inserted into pBI121, a plant overexpression vector through homologous recombination. The GbVIP1 was expressed by 35S promoter and selected by NPTII. The conducted pBI121–GbVIP1 vector was transformed into Agrobacterium strain GV3101. For Agrobacterium-mediated transformation, sterile leaves derived from tissue-cultured tobacco plants were cut into 1 cm² squares. The leaf explants were pre-cultured on MS solid medium for 3 days in darkness. Agrobacterium tumefaciens harboring the pBI121–GbVIP1 vector was cultured at 28 °C overnight and when the OD₆₀₀ value reached 0.6, Agrobacterium solution was harvested using a centrifuge (3 500 r·min^-1, 10 min) and re-suspended in MS liquid medium. The pre-cultured tobacco leaves were placed in Agrobacterium suspensions for 20 min, dried in sterile filter paper, and then co-cultured on solid MS medium at 25 °C in darkness for 2 days. The infected leaf disks were transferred to the selection medium (MS solid medium containing 300 mg·L^-1 carbenicillin, 100 mg·L^-1 cefotaxime, 100 mg·L^-1 kanamycin, 1.0 mg·L^-1 6-BA and 0.1 mg·L^-1 IAA) at 25 °C in light conditions for several days until putative transgenic shoots were regenerated. Selection medium was changed every 2 weeks. The regenerated shoots were transferred into rooting medium (MS solid medium containing 300 mg·L^-1 carbenicillin, 100 mg·L^-1 cefotaxime and 100 mg·L^-1 kanamycin) for taking roots. Transgenic detection was performed using PCR and real-time PCR.

Morbidity situation analysis

The DI was used to measure the morbidity situation of cotton seedlings after V. dahliae infection. A higher DI value indicates less, disease resistance. According to leaf chlorosis symptoms, cotton seedlings were classified into five grades: 0 (healthy plants), 1 (25% of leaves showing infection symptoms), 2 (25%∼50% of leaves showing symptoms), 3 (50%∼75% of leaves showing symptoms) and 4 (more than 75% of leaves showing symptoms) (Zhang et al. 2012a). The DI was calculated using the following formula:

$$ DI=\frac{\sum number\ of\ infected\ plant\times Disease\ grade}{total\ number\ of\ infected\ plant\times 4}\times 100\% $$

The incidence of detached tobacco leaves was investigated at 16 days after inoculation with V. dahliae. Disease grade was according to 0–3 scale: 0 (healthy leaves), 1 (=10%∼20% leaf area showing infection symptoms), 2 (=20%∼50% leaf area showing symptoms), and 3 (= > 50% leaf area showing symptoms).

Trypan blue test

Trypan is a kind of cell dye that stains dead cells blue. Leaves from infected cotton seedlings were dipped in trypan blue dye solution containing 15 mg of trypan blue, 10 mL of 85% lactic acid, 10 mL of glycerol, 10 mL of phenol, 10 mL of sterile water for 15 min in vacuum conditions. Then the leaves were placed in boiling water for 10 min in order to fix the dye. Finally, 2.5 g·mL^-1 chloral hydrate solution was used to decolorize (Choi and Hwang 2011).

qRT-PCR

Tissue-specific expression of VIP1 and its differential expression patterns in different conditions were investigated using qRT-PCR. SYBR Primix Ex Taq™ II (Tli RNaseH Plus), Bulk (TaKaRa) were used for qRT-PCR and a 20 μL reaction volume including 10 μL 2× SYBR Premix Ex Taq II, 2 μL of cDNA template, 0.8 μL of PCR forward primer (10 μmol·L^-1), 0.8 μL of PCR reverse primer (10 μmol·L^-1), 0.4 μL of ROX and 6 μL of sterile water was used. The qRT-PCR was performed on an ABI 7500 qRT-PCR System (Applied Biosystems). The qRT-PCR procedure consisted of 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 34 s. The dissociation curves of each reaction were checked and all reactions were performed with three biological replicates. Cotton and tobacco actin genes were used as an internal control for normalization of expression values. Results of qRT-PCR were calculated by 2^−ΔΔC_t method (Livak and Schmittgen 2001) and statistical analysis of qRT-PCR results was conducted using DPS software (IBM, USA). All primers used in qRT-PCR were synthesized by GENEWIZ and are listed in Additional file 3.

Bioinformatics analysis

The BLAST tool in the National Center for Biotechnology Information website was used to find VIP1 genes from different plants. BioEdit software was applied for alignment of nucleotide and protein sequences. Conserved domains were analyzed using WebLogo software and CD-Search software. Mega 6.0 software was used to construct the phylogenetic tree.