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Plant Cell Reports https://doi.org/10.1007/s00299-018-2294-5

ORIGINAL ARTICLE

A novel VIGS method by agroinoculation of cotton seeds and application for elucidating functions of GhBI-1 in salt-stress response

Jingxia Zhang1 · Furong Wang1 · Chuanyun Zhang1 · Junhao Zhang3 · Yu Chen1 · Guodong Liu1 · Yanxiu Zhao4 · Fushun Hao2 · Jun Zhang1,4

Received: 3 January 2018 / Accepted: 10 May 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018

AbstractKey message A VIGS method by agroinoculation of cotton seeds was developed for gene silencing in young seedlings and roots, and applied in functional analysis of GhBI-1 in response to salt stress.Abstract Virus-induced gene silencing (VIGS) has been widely used to investigate the functions of genes expressed in mature leaves, but not yet in young seedlings or roots of cotton (Gossypium hirsutum L.). Here, we developed a simple and effective VIGS method for silencing genes in young cotton seedlings and roots by soaking naked seeds in Agrobacterium cultures carrying tobacco rattle virus (TRV)-VIGS vectors. When the naked seeds were soaked in Agrobacterium cultures with an OD600 of 1.5 for 90 min, it was optimal for silencing genes effectively in young seedlings as clear photo-bleaching pheno-type in the newly emerging leaves of pTRV:GhCLA1 seedlings were observed at 12–14 days post inoculation. Silencing of GhPGF (cotton pigment gland formation) by this method resulted in a 90% decrease in transcript abundances of the gene in roots at the early development stage. We further used the tool to investigate function of GhBI-1 (cotton Bax inhibitor-1) gene in response to salt stress and demonstrated that GhBI-1 might play a protective role under salt stress by suppressing stress-induced cell death in cotton. Our results showed that the newly established VIGS method is a powerful tool for elucidating functions of genes in cotton, especially the genes expressed in young seedlings and roots.

Keywords Seed soak agroinoculation VIGS (SSA-VIGS) · Cotton · Bax inhibitor-1 · Salt tolerance

Introduction

Cotton (Gossypium hirsutum L.) is one of the most impor-tant natural fiber crops in the world. It is also the preferred main crop in saline land for its high salinity tolerance. The key traits of cotton such as yield, fiber quality and stress tolerances are predominantly controlled by multiple genes (Gunapati et al. 2016; Liang et al. 2017; Qin et al. 2017; Ullah et al. 2018; Wu et al. 2018; You et al. 2017; Zhang et al. 2017a, b; Zhao et al. 2015a, b). Therefore, it is essential

Jingxia Zhang and Furong Wang contributed equally to this work.

Communicated by Chun-Hai Dong.

Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s0029 9-018-2294-5) contains supplementary material, which is available to authorized users.

* Fushun Hao haofsh@henu.edu.cn

* Jun Zhang scrczj@saas.ac.cn; zj0928@126.com

1 Key Laboratory of Cotton Breeding and Cultivation in Huang-Huai-Hai Plain, Ministry of Agriculture, Cotton Research Center of Shandong Academy of Agricultural Sciences, Jinan 250100, China

2 State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, College of Life Science, Henan University, Kaifeng 475004, China

3 Nanjing Agricultural University, Nanjing 210095, China4 College of Life Science, Shandong Normal University,

Jinan 250014, China

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for researchers to dissect roles and functional mechanisms of these valuable genes in cotton.

In recent years, whole genomes of diploid and tetraploid cotton species have been sequenced along with the rapid development of sequencing technologies (Li et al. 2014; Paterson et al. 2012; Zhao et al. 2015a, b), which provide researchers a great opportunity to explore the functions and mechanisms of genes regulating the main agronomic traits in cotton. It is known that reverse genetics methods, such as RNAi (RNA interference) and T-DNA insertional mutagenesis, are powerful tools for studying gene functions in various plant species. In the methods, effective genetic transformation is required for generating stable transgenic lines. Despite great achievements in cotton genetic trans-formation, there still are some serious shortcomings, such as genotype dependency, low transformation efficiency and time-consuming procedures, which markedly limit the pro-gress of gene function study in cotton.

Virus-induced gene silencing (VIGS) is an alternative reverse genetics method for investigating gene functions in plants. It is established on the basis of RNA-mediated post-transcriptional gene silencing that plays roles as an antivirus defense system in plants (Hamilton and Baulcombe 1999). As a tool, VIGS has many advantages over conventional techniques, such as easy manipulation, high effectiveness, independent of genetic transformation, suitable for large-scale functional analysis of genes and for analyzing genes causing lethal phenotypes, and so on (Burch-Smith et al. 2004; Dinesh-Kumar et al. 2003; Lu et al. 2003; Ramegowda et al. 2014).

In cotton, VIGS system is first developed from gemini-virus Cotton leaf crumple virus (CLCrV), and a gene gun is needed to deliver biolistic particles (Tuttle et al. 2008). Later, Gao et al. (2011) successfully performed an Agro-bacterium-mediated VIGS assay using tobacco rattle virus (TRV) vectors in leaves of diverse cotton cultivars. In the assay, inoculation was carried out by hand-infiltrating the Agrobacturium cultures carrying TRV-VIGS vectors into cotton cotyledons (Gao et al. 2011). Further, a vacuum infil-tration method was used in VIGS experiments to investigate gene functions during cotton fiber development (Qu et al. 2012). To date, although various VIGS systems have been proved to work in cotton, the utilization of the current VIGS methods is limited in elucidating the functions of genes in leaves of mature plants and reproductive organs such as fib-ers. It was not known whether these methods can be used to assess functions of genes in very young seedlings and roots. Hence, it is urgently required to improve VIGS technology to its full potential utility in young seedlings and roots in cotton.

Bax inhibitor-1 (BI-1), first identified as a suppressor of Bax-induced cell death, is a highly evolutionarily conserved cell death regulator. BI-1-like genes have also been found

and studied in diverse plants (Isbat et al. 2009; Kawai-Yam-ada et al. 2001, 2004; Wang et al. 2012; Xu and Reed 1998; Zha et al. 1996). In Arabidopsis, the AtBI-1 gene encodes a protective protein induced by various stress and modu-lates the activation of stress-induced programmed cell death (PCD) (Watanabe and Lam 2009). In our previous work, a homologous gene of AtBI-1 was cloned from a cDNA library of cotton root under salt stress and was named GhBI-1. The expression level of GhBI-1 was elevated after salt stress (data not published), indicating that GhBI-1 may be involved in response to salt stress. However, it is unclear whether and how GhBI-1 functions in salt tolerance and salt-induced cell death in cotton.

In this study, a simple seed soak agroinoculation VIGS (SSA-VIGS) method is developed to elicit gene silencing in young cotton seedlings and roots. Moreover, silencing of GhBI-1 compromised salt tolerance of young cotton seed-lings and accelerated the salt-stress-induced cell death in roots. The SSA-VIGS method will paves an important way for functional analysis of genes in young seedlings and roots in cotton.

Materials and methods

Plant materials and growth conditions

TM-1 and LMY37 cotton plants were used in the experi-ments. LMY37 is an elite cultivar with high salinity toler-ance developed by our team. For VIGS experiments, both TM-1 and LMY37 seedlings were grown in a growth cham-ber with a 14 h light/10 h dark cycle at 25–27 °C.

SSA‑VIGS procedure

The pTRV-VIGS vectors were constructed as described pre-viously (Gao et al. 2011). Briefly, cDNA fragments of cotton Cloroplastos alterados 1 (GhCLA1, 420 bp), Pigment gland formation (GhPGF, 398 bp) and Bax inhibitor 1 (GhBI-1, 433 bp) were amplified from TM-1 by PCR with gene spe-cific primers (listed in Supplemental data 1). The resulting products were cloned into pTRV2 with EcoRI and KpnI to produce recombinant vectors pTRV:GhCLA1, pTRV:GhPGF and pTRV:GhBI-1, respectively. These recombinant vectors and the empty vector (pTRV:00) were then introduced into the Agrobacterium strain GV3101, and resuspended in the solutions (10 mM 3-(N-morpholino) ethane sulfonic acid, 10 mM MgCl2, 200 µM acetosyringone (AS), and 5% (w/v) sucrose). The densities of the solutions were set as OD val-ues of 0.5, 1.5, and 2.5 at 600 nm as reported previously (Kim et al. 2016; Zhang et al. 2017a, b). Then, Agrobacte-rium cultures containing pTRV1 and pTRV2 or its deriva-tives were mixed in 1:1 ratios.

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Cotton seeds were surface-sterilized with 3% hydro-gen peroxide solution for 60 min and washed for several times with sterile ddH2O. Then, the seeds were immersed into sterile ddH2O for 4–6 h at 28 °C. Seed coats were then totally removed. The naked seeds were dipped into different concentrations of Agrobacterium suspensions for different durations. The inoculated seeds were cultured in Murashige and Skoog (MS) liquid medium with 200 µM AS at 26 °C in dark for 2 d, and then sowed on MS solid medium with 60–100 µg mL− 1 rifampicin antibiotics. After recovery for 1–2 days, the germinated seeds were planted into nutrition soil and grown in the growth chamber. Gene silencing effi-ciency of GhCLA1 was estimated by calculating percent-ages of the seedlings showing photo bleaching phenotypes in all seedlings inoculated with pTRV:GhCLA1 vectors in the experiments.

Total RNA extraction and cDNA synthesis

Total RNA was extracted from leaves and roots using TRIzol reagent (Invitrogen, http://www.invit rogen .com) following the manufacturer’s instructions. RNA quality was checked on 1.0% (w/v) agarose gels. The cDNA was synthesized from the RNA using a PrimeScript RT reagent kit with gDNA eraser (TaKaRa).

Measurement of transcript abundances by qRT‑PCR

The amount of RNA in each reaction was normalized using cotton Actin9 (GhActin9) and cotton Histone3 (GhHis3) (Wang et al. 2013). Gene-specific primers were designed based on the gene sequences using the Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA) and listed in Table S1. Each 25 µL reaction sample was run on a Bio-Rad IQ2 sequence detection system with the Applied Biosystems software. The relative expression level of each gene was calculated using the 2−∆∆Ct method. In all cases, the relative expression level of a target gene is presented as its transcriptional level in the potentially silenced sample relative to that in the sample inoculated with the empty vector.

Salt tolerance analysis

Seedlings were treated with high salt stress (300 mM NaCl) for indicated time periods in the growth chamber since the concentration of NaCl can produce harsh salt stress in cotton plants (Wei et al. 2017). Then, relative water content (RWC) was investigated. Briefly, leaves and roots were harvested and weighed (fresh weight, FW), and soaked in distilled water for 4 h and weighted (saturated fresh weight, SFW). The samples were killed out at 105 °C for 3 h and stoved at 80  °C overnight. The remaining

weight was the dry weight (DW). RWC was calculated according to the formule: RWC (%) = [(FW − DW)/(SFW − DW)] × 100. Cell death was detected by trypan blue stain as described (Leite et al. 1999). In brief, trypan blue was dissolved in ddH2O at a final concentration of 10 mg mL− 1. The roots were immersed in the solution and incubated for 30 min at room temperature and then washed with ddH2O for at least three times.

DNA isolation and electrophoresis

To detected PCD, a DNA smearing assay was performed. About 100 mg root tips (< 2 cm in length) were collected after treatment with 300 mM NaCl at indicated time points. Isolation of DNA was carried out as reported (Chen et al. 2014). To visualize DNA smearing, 8 µg genomic DNA for each sample was run on a 1.5% agarose (w: v) gel at a constant voltage of 70 V for 120 min. The experiment was repeated three times.

Results

GhCLA1 was silenced in young seedlings by SSA‑VIGS

To test whether gene silencing can be induced in young cot-ton seedlings by SSA-VIGS method, we selected endogenous gene GhCLA1, which is involved in the modulation of chlo-roplast developments, as a visual marker in this study (Gao et al. 2011). The pTRV:GhCLA1 recombinant vectors were constructed (Fig. 1a) and introduced into Agrobacterium. Germinating seeds were inoculated with the Agrobacterium cultures containing the recombinant pTRV:GhCLA1 vector or empty vector (pTRV:00, the control) for 90 min. Then, the inoculated seeds were grown in a growth chamber. Numer-ous newly emerging photo-bleached leaves were observed in the pTRV:GhCLA1 seedlings at 12–14 days post inoculation (dpi) and the albino plants became severe stunted by the late growth stage (35 days old). On the contrary, the vector control plants did not show any photobleaching phenotype and grew normally (Fig. 1a, b). The chlorophyll contents of leaves from pTRV:GhCLA1 seedlings were also markedly reduced compared with those of the controls (Fig. 1c). Fur-ther, the transcriptional levels of GhCLA1 in pTRV:GhCLA1 or vector control seedlings were measured by quantitative real-time PCR (qRT-PCR). The transcript abundances of GhCLA1 in pTRV:GhCLA1 plants were reduced to 10 and 17% of those in the control at 12 dpi and 35 dpi (Fig. 1d). These results indicate that silencing of endogenous genes in cotton can be triggered by SSA-VIGS method.

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Optimizations of SSA‑VIGS

To optimize the conditions for effective gene silencing by SSA-VIGS, some pivotal inoculation parameters including host explant type (seed type), soaking duration and Agro-bacterium density were assessed. The impacts of differ-ent explants (germinating seeds with or without coats) on silencing efficiency were detected in the initial experiments. The results showed that the silencing efficiency of GhCLA1 employing germinating seeds without coats (naked seeds) as explants was about seven times higher than that using seeds with coats (Fig. 2). Therefore, naked seeds were used as preferred host explants in the following experiments. Next, effects of inoculation duration and Agrobacterium concentration on seed germination rate (after inoculation) as well as silencing efficiency were investigated. As shown in Table 1, treatments with prolonged inoculation time and higher Agrobacterium culture concentrations improved the silencing efficiency of GhCLA1, however, decreased the ger-mination rates of the inoculated seeds. Manipulations with extreme high Agrobacterium culture concentration (the final OD 600 of 2.5) or too long inoculation time (120 min) could not elevate the silencing efficiency of genes (Table 1). Taken together, naked seeds soaked in Agrobacterium cultures with a final OD 600 of 1.5 for 90 min was optimal for SSA-VIGS, 90% of seeds was germinated and 73.33% of photo-bleach-ing plants was produced (Table 1).

GhPGF was silenced in roots by SSA‑VIGS

To ascertain whether gene silencing can be induced in roots using SSA-VIGS method, we select GhPGF (cotton pig-ment gland formation gene), highly expressed in roots and leaves. GhPGF is a positive regulator in gland formation

Fig. 1 Silencing of GhCLA1 by SSA-VIGS method in cotton. a Con-struction of the pTRV:GhCLA1 VIGS vectors. b The photo-bleaching phenotypes of cotton seedlings inoculated with pTRV:GhCLA1 and empty vectors at 12 dpi (left) and 35 dpi (right). c The chlorophyll

contents of leaves from seedlings as described in (b). d Relative GhCLA1 transcript levels in leaves of pTRV:GhCLA1 or the control plants. GhActin 9 was used as an internal control. Each bar value rep-resents mean ± SD (n = 6) of three independent experiments

Fig. 2 Effects of seed type on gene silencing efficiency. Left column reveals the silencing efficiency of GhCLA1 by inoculating seeds with coats (as presented below) with pTRV:GhCLA1; right column shows the silencing efficiency of GhCLA1 by inoculating naked seeds (as presented below) with pTRV:GhCLA1. Each bar value represents mean ± SD (n = 20) of six independent experiments

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in cotton. The silencing of GhPGF causes a clear gland-less phenotype in leaves and shoots, which could be used as a visible marker in the VIGS study (Ma et al. 2016). Plants infiltrated with Agrobacterium cultures containing pTRV:GhPGF or empty vectors were obtained. The accumu-lation of virus in the pTRV:GhPGF roots was measured by PCR with specific primers of pTRV2 coat protein gene. The expected PCR products (289 bp) were detected as early as 5 dpi (Fig. 3a). Then we measured the transcript abundances of GhPGF in roots. As shown in Fig. 3b, the expression levels of GhPGF in pTRV:GhPGF roots were prominently lower than those in the control. In particular, the transcript abundances of GhPGF in pTRV:GhPGF plants decreased to the minimum level, only about 10% of those in the control at 15 dpi (Fig. 3b). Compared with that in the control, GhPGF transcript levels in pTRV:GhPGF roots were reduced from 10 to 28 dpi. As expected, the leaves and shoots from pTRV:GhPGF seedlings showed obvious glandless pheno-type at 12 dpi (Fig. 3c) and the GhPGF transcript levels in pTRV:GhPGF plants were markedly reduced compared to that in the control (Fig. 3d). To confirm the expression pat-terns of GhPGF in VIGS treated plants, we used GhHis3 as another reference gene for qRT-PCR experiments and got the similar results (Fig. S1). Taken together, the data hint that SSA-VIGS can be used to elicit gene silencing in roots, as well as in leaves and shoots at early development stage of cotton.

Silencing of GhBI‑1 by SSA‑VIGS decreased salt tolerance in cotton seedlings

We further explore the functions of GhBI-1 under salt stress using the SSA-VIGS method. Cotton plants

inoculated with Agrobacterium cultures carrying pTRV:GhBI-1 or empty vectors were generated and no sig-nificant phenotypic differences were observed between the pTRV:GhBI-1 and the control plants under normal condi-tions (data not shown). The expression levels of GhBI-1 in pTRV:GhBI-1 seedlings were dramatically reduced compared with those in the control at 20 dpi (Fig. 4a). The qRT-PCR results were also confirmed by GhHis3 and showed the similar results (Fig. S2).

The possible roles of GhBI-1 in salt tolerance were also evaluated. As shown in Fig. 4b, the pTRV:GhBI-1 seed-lings were more sensitive to salt stress compared with the controls. Clear drooping and wilting symptoms occurred in pTRV:GhBI-1 seedlings after treatment with 300 mM NaCl for 1 days. By contrast, the control seedlings did not show obvious stress symptom. After exposure to salt stress for 4 d, pTRV:GhBI-1 seedlings showed extreme severe stress symptom (such as dried up shoots), which did not occur in control plants (Fig. 4b). The leaf RWC of pTRV:GhBI-1 seedlings was also decreased compared with that of the controls (Fig. 4c). In addition, cell death was investigated by trypan blue stain after constant and harsh salt stress (300 mM NaCl treated for 3d). Clearly, the pTRV:GhBI-1 leaves stained deeper than the controls (Fig. 4d).

It is well known that the root is the first organ directly fac-ing salt stress. We, therefore, investigated the role of GhBI-1 in inhibition of cell death caused by salt stress in young cotton roots. As shown in Fig. 4d, the pTRV:GhBI-1 roots were stained deeper than those of the controls by trypan blue. Besides, the cell death rates of the pTRV:GhBI-1 root tips were prominently higher than those of the controls. After treatment with 300 mM NaCl for 14 h, the death rate of pTRV:GhBI-1 roots was 24.7% of the control (Fig. 4e).

Table 1 Effectiveness of GhCLA1 silencing in cotton inoculated with different soak durations and concentrations of Agrobacterium cultures

a Efficiency (%) = Number of seeds showing the photo-bleaching phenotype/total number of seeds infected with Agrobacterium suspension × 100. Mean values of three independent experiments (±) with SD are shown

Serial no. Soak inocula-tion duration (min)

Concentra-tion (OD 600)

No. of seeds inoculated

Mean no. of seeds germi-nated

Mean no. of seedlings bleached

Efficiencya (%)

1 30 0.5 50 50.00 7 ± 1.00 14.00 ± 2.002 60 0.5 50 47.67 ± 2.08 11.33 ± 1.53 22.67 ± 3.063 90 0.5 50 45.67 ± 1.08 14.33 ± 1.15 28.67 ± 2.314 120 0.5 50 39.67 ± 3.51 15.66 ± 2.89 31.33 ± 5.775 30 1.5 50 49.67 ± 0.58 26.33 ± 3.06 52.67 ± 6.116 60 1.5 50 46.00 ± 1.00 30.66 ± 1.53 61.33 ± 3.067 90 1.5 50 45.00 ± 1.73 36.67 ± 1.53 73.33 ± 3.068 120 1.5 50 29.33 ± 1.53 18.00 ± 2.65 36.00 ± 5.299 30 2.5 50 40.00 ± 1.00 9.00 ± 4.58 18.00 ± 9.1710 60 2.5 50 28.67 ± 5.51 14.00 ± 2.00 28.00 ± 4.0011 90 2.5 50 18.00 ± 3.00 11.00 ± 3.00 22.00 ± 6.0012 120 2.5 50 12.33 ± 6.51 8.33 ± 2.08 16.67 ± 4.16

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Taken together, these data suggest that GhBI-1 plays an important role in resistance to salt stress in cotton.

Silencing of GhBI‑1 promoted salt‑induced PCD

To determine whether GhBI-1 is able to regulate salt-stress-induced PCD in roots, degradation of DNA, a diagnostic marker for PCD, was investigated. Genomic DNA was extracted from root tips of the pTRV:GhBI-1 and control seedlings after treatment with 300  mM NaCl for indi-cated time points, and then separated by electrophoresis. The degree of DNA smearing was remarkably accelerated in pTRV:GhBI-1 roots compared with that in the control (Fig. 5), suggesting that GhBI-1 is involved in the modula-tion of PCD progression under severe salt stress.

Discussion

VIGS is an efficient strategy for functional analysis of targeted genes in plants. The increased virus vectors and improved procedures for VIGS broaden the applications of this technology (Constantin et al. 2004; Ryu et al. 2004;

Tuttle et al. 2012; Valentine et al. 2004; Zhang et al. 2017a, b). For example, agrodrench, the modified VIGS agroinocu-lation method, was reported to be more efficient for defining the roles of genes in roots of diverse Solanaceae species. There, soils adjacent to roots of the plants were soaked with Agrobacterium suspension carrying the pTRV-VIGS vec-tors, and gene silencing occurred 2–3 weeks earlier than those done with the inoculation of leaf infiltration (Ryu et al. 2004). Very recently, Zhang et al. (2017a, b) described a novel VIGS method via vacuum and co-cultivation agroinfil-tration of germinated seeds in wheat and maize, which could elicit whole-plant level VIGS. In this study, we developed a SSA-VIGS method in cotton. The SSA-VIGS experiment was carried out by soaking naked germinating seeds in Agro-bacterium cultures containing TRV-VIGS vectors to elicit gene silencing in leaves and roots at the early development stage (Figs. 1, 3). The technology was easily manipulated, and the gene silencing efficiency was high. More impor-tantly, it enables us to readily study the functions of target genes expressed in very young seedlings and roots in cotton.

VIGS efficiency is affected by many factors, for instance virus vectors, host explants, Agrobacterium culture con-centrations and infection time (Tuttle et  al. 2012). We

Fig. 3 Silencing of GhPGF by SSA-VIGS method in cotton seed-lings. a The accumulations of virus in roots of pTRV:GhPGF plants measured by PCR. The arrow indicates the predicted PCR products (289 bp). b Relative GhPGF transcript levels in roots of pTRV:GhPGF or vector control plants at different time points after inoculation. c The phenotypes of leaves and shoots from

pTRV:GhPGF or vector control plants at 15 dpi. The arrows indi-cate the darkly pigmented glands in GhPGF silenced leaf and shoot. d Relative transcript levels of GhPGF in leaves and shoots of pTRV:GhPGF or vector control plants at 15 dpi. GhActin 9 was used as an internal control. Each bar value represents mean ± SD (n = 6) of three independent experiments

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demonstrated that removing coats of cotton seeds clearly increased the silencing efficiency of GhCLA1 by SSA-VIGS (Fig. 2), probably due to easier entrance of Agrobacterium in the naked seeds than in the seeds with coats. Similar results are also observed in maize (Zhang et al. 2017a, b). In addi-tion, Zhang et al. (2017a, b) found it is optimal for TRV-VIGS that agroinfiltration of germinated wheat seeds and cut maize seeds with the Agrobacterium cultures with a final OD 600 of 0.3 by vacuuming for different times followed by co-cultivation for 15 h. In the present study, 90 min of soak-ing inoculation with the Agrobacterium cultures with a final OD 600 of 1.5 led to an ideal silencing efficiency for SSA-VIGS (Table 1). Prolonged soak time increased the silenc-ing efficiency but decreased the survival rate of inoculated seeds (Table 1). Besides, too many Agrobacterium adversely impacted the seed survival whereas too few Agrobacterium resulted in lower silencing efficiency. Hence, it seems that the optimal inoculation procedure for VIGS may highly depend on plant species, tissues and organs in plants.

BI-1 has been addressed to inhibit cell death caused by biotic or abiotic stress such as heat, cold and drought stresses in plants (Duan et al. 2010; Eichmann et al. 2004; Nagano et al. 2014; Wang et al. 2012; Watanabe and Lam 2008). For example, the Arabidopsis atbi-1 mutant plants display increased sensitivity in root elongation and accelerated cell death in root tips under drought stress (Duan et al. 2010). In the present study, decreased expression of GhBI-1 by SSA-VIGS resulted in reduced RWC and accelerated cell death rate in root tips under high salt conditions, implying that GhBI-1 is of quite importance in cotton response to salinity stress, and SSA-VIGS is helpful for functional analysis of genes in cotton (Fig. 4c–e). These results also suggest that plant BI-1 may have more general roles in suppression of cell death under stress conditions.

PCD plays important roles in plant development and adaptation to various biotic and abiotic stresses (Barany et al. 2018; Cao and Li 2010; Duan et al. 2010; Mao et al. 2018). In Arabidopsis, PCD appears to regulate the developmental

Fig. 4 Functional analysis of GhBI-1 under salt stress. a Relative transcript levels of GhBI-1 of plants inoculated with pTRV:GhBI-1 or empty vector control. b The pheno-types of the young seedlings inoculated with pTRV:GhBI-1 or empty vector under salt stress (300 mM NaCl). c The RWC of leaves from pTRV:GhBI-1 or vector control plants under salt stress. d Cell death in leaves and roots from pTRV:GhBI-1 or vector control plants detected by trypan blue staining after exposure to 300 mM NaCl for 3 days. e The rates of cell death in root tips from pTRV:GhBI-1 or vector control plants under salt stress. Each bar value represents mean ± SD (n = 6) of three independent experiments

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plasticity of root system architecture and subsequent adap-tation to severe water stress, and AtBI-1 plays a key role in the process (Duan et al. 2010). Here, we provide evidences that salt-stress-induced PCD occurred in cotton root tips and GhBI-1 functions as an attenuator of PCD under severe salt-stress conditions (Fig. 5). However, we did not detect the DNA ladder, the standard marker of PCD, in root tips. The possible reason may be that DNA fragments generated in the cells undergoing PCD were too small to be detected by electrophoresis (Gunawardena et al. 2004). Similar results were found in previous studies (Duan et al. 2010; Liu et al. 2010). Further studies are needed to better understand the nature and mechanisms of PCD regulated by GhBI-1 under salt stress.

In conclusion, the SSA-VIGS technology established in this report had some significant advantages: suitability for investigating the functions of genes in very young seedlings and roots, greater simplicity and rapidity. Its application will effectively promote the development of gene functional analysis in cotton and other plants.

Author contribution statement JXZ and FW performed the experiments; YC, CZ, GL and JHZ analyzed data and

prepared figures; JXZ and FW prepared the preliminary manuscript; JZ, FH and YXZ designed the experiments and corrected the manuscript.

Acknowledgements The authors thank professor Libo Shan, Texas A&M University, and professor Zhaohu Li, China Agricultural Uni-versity, for providing the pTRV-VIGS vectors. This work is supported by the National Science Foundation in China [31501351]; Youth Sci-entific Research Foundation of Shandong Academy of Agricultural Science [2015YQN04]; the National Project of Modern Agricultural Industry Technology System in China [CARS-15-05]; the Taishan Scholars Program of Shandong Province [ts201511070] and the State Key Laboratory of Cotton Biology Open Fund.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

References

Barany I, Berenguer E, Solis MT, Perez-Perez Y, Santamaria ME, Crespo JL, Risueno MC, Diaz I, Testillano PS (2018) Autophagy is activated and involved in cell death with participation of cath-epsins during stress-induced microspore embryogenesis in barley. J Exp Bot 69:1387–1402

Burch-Smith TM, Anderson JC, Martin GB, Dinesh-Kumar SP (2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J 39:734–746

Cao M, Li X (2010) Die for living better: plants modify root system architecture through inducing PCD in root meristem under severe water stress. Plant Signal Behav 5:1645–1646

Chen Y, Chen X, Wang H, Bao Y, Zhang W (2014) Examination of the leaf proteome during flooding stress and the induction of pro-grammed cell death in maize. Proteome Sci 12:33

Constantin GD, Krath BN, MacFarlane SA, Nicolaisen M, Johansen IE, Lund OS (2004) Virus-induced gene silencing as a tool for functional genomics in a legume species. Plant J 40:622–631

Dinesh-Kumar SP, Anandalakshmi R, Marathe R, Schiff M, Liu Y (2003) Virus-induced gene silencing. In: Grotewold E (ed) Plant functional genomics. Humana Press, Totowa, pp 287–293

Duan Y, Zhang W, Li B, Wang Y, Li K, Han SC, Zhang Y, Li X (2010) An endoplasmic reticulum response pathway mediates pro-grammed cell death of root tip induced by water stress in Arabi-dopsis. New Phytol 186:681–695

Eichmann R, Schultheiss H, Kogel KH, Huckelhoven R (2004) The barley apoptosis suppressor homologue BAX inhibitor-1 compro-mises nonhost penetration resistance of barley to the inappropri-ate pathogen Blumeria graminis f. sp. tritici. Mol Plant Microbe Interact 17:484–490

Gao X, Wheeler T, Li Z, Kenerley CM, He P, Shan L (2011) Silenc-ing GhNDR1 and GhMKK2 compromises cotton resistance to Verticillium wilt. Plant J 66:293–305

Gunapati S, Naresh R, Ranjan S, Nigam D, Hans A, Verma PC, Gadre R, Pathre UV, Sane AP, Sane VA (2016) Expression of GhNAC2 from G. herbaceum, improves root growth and imparts tolerance to drought in transgenic cotton and Arabidopsis. Sci Rep 6:24978

Gunawardena AH, Greenwood JS, Dengler NG (2004) Programmed cell death remodels lace plant leaf shape during development. Plant Cell 16:60–73

Fig. 5 DNA smearing induced by severe salt stress in roots

Plant Cell Reports

1 3

Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950–952

Isbat M, Zeba N, Kim SR, Hong CB (2009) A BAX inhibitor-1 gene in Capsicum annuum is induced under various abiotic stresses and endows multi-tolerance in transgenic tobacco. J Plant Phys-iol 166:1685–1693

Kawai-Yamada M, Jin L, Yoshinaga K, Hirata A, Uchimiya H (2001) Mammalian Bax-induced plant cell death can be down-regu-lated by overexpression of Arabidopsis Bax Inhibitor-1 (AtBI-1). Proc Natl Acad Sci USA 98:12295–12300

Kawai-Yamada M, Ohori Y, Uchimiya H (2004) Dissection of Arabi-dopsis Bax inhibitor-1 suppressing Bax-, hydrogen peroxide-, and salicylic acid-induced cell death. Plant Cell 16:21–32

Kim KH, Lim S, Kang YJ, Yoon MY, Nam M, Jun TH, Seo MJ, Baek SB, Lee JH, Moon JK, Lee SH, Lee SH, Lim HS, Moon JS, Park CH (2016) Optimization of a virus-induced gene silenc-ing system with soybean yellow common mosaic virus for gene function studies in soybeans. Plant Pathol J 32:112–122

Leite M, Quinta-Costa M, Leite PS, Guimaraes JE (1999) Critical evaluation of techniques to detect and measure cell death—study in a model of UV radiation of the leukaemic cell line HL60. Anal Cell Pathol 19:139–151

Li F, Fan G, Wang K, Sun F, Yuan Y, Song G, Li Q, Ma Z, Lu C, Zou C, Chen W, Liang X, Shang H, Liu W, Shi C, Xiao G, Gou C, Ye W, Xu X, Zhang X, Wei H, Li Z, Zhang G, Wang J, Liu K, Kohel RJ, Percy RG, Yu JZ, Zhu YX, Wang J, Yu S (2014) Genome sequence of the cultivated cotton Gossypium arboreum. Nat Genet 46:567–572

Liang C, Liu Y, Li Y, Meng Z, Yan R, Zhu T, Wang Y, Kang S, Ali Abid M, Malik W, Sun G, Guo S, Zhang R (2017) Activation of ABA receptors gene GhPYL9-11A is positively correlated with cotton drought tolerance in transgenic arabidopsis. Front Plant Sci 8:1453

Liu WZ, Zhou YF, Wang X, Jiao ZJ (2010) Programmed cell death during pigment gland formation in Gossypium hirsutum leaves. Plant Biol (Stuttg) 12:895–902

Lu R, Martin-Hernandez AM, Peart JR, Malcuit I, Baulcombe DC (2003) Virus-induced gene silencing in plants. Methods 30:296–303

Ma D, Hu Y, Yang C, Liu B, Fang L, Wan Q, Liang W, Mei G, Wang L, Wang H, Ding L, Dong C, Pan M, Chen J, Wang S, Chen S, Cai C, Zhu X, Guan X, Zhou B, Zhu S, Wang J, Guo W, Chen X, Zhang T (2016) Genetic basis for glandular trichome forma-tion in cotton. Nat Commun 7:10456

Mao C, Ding J, Zhang B, Xi D, Ming F (2018) OsNAC2 positively affects salt-induced cell death and binds to the OsAP37 and OsCOX11 promoters. Plant J 94:454–468

Nagano M, Ishikawa T, Ogawa Y, Iwabuchi M, Nakasone A, Shi-mamoto K, Uchimiya H, Kawai-Yamada M (2014) Arabidopsis Bax inhibitor-1 promotes sphingolipid synthesis during cold stress by interacting with ceramide-modifying enzymes. Planta 240:77–89

Paterson AH, Wendel JF, Gundlach H, Guo H, Jenkins J, Jin D, Llewellyn D, Showmaker KC, Shu S, Udall J, Yoo MJ, Byers R, Chen W, Doron-Faigenboim A, Duke MV, Gong L, Grimwood J, Grover C, Grupp K, Hu G, Lee TH, Li J, Lin L, Liu T, Marler BS, Page JT, Roberts AW, Romanel E, Sanders WS, Szadkowski E, Tan X, Tang H, Xu C, Wang J, Wang Z, Zhang D, Zhang L, Ashrafi H, Bedon F, Bowers JE, Brubaker CL, Chee PW, Das S, Gingle AR, Haigler CH, Harker D, Hoffmann LV, Hovav R, Jones DC, Lemke C, Mansoor S, ur Rahman M, Rainville LN, Ram-bani A, Reddy UK, Rong JK, Saranga Y, Scheffler BE, Scheffler JA, Stelly DM, Triplett BA, Van Deynze A, Vaslin MF, Wagh-mare VN, Walford SA, Wright RJ, Zaki EA, Zhang T, Dennis ES, Mayer KF, Peterson DG, Rokhsar DS, Wang X, Schmutz J

(2012) Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres. Nature 492:423–427

Qin LX, Chen Y, Zeng W, Li Y, Gao L, Li DD, Bacic A, Xu WL, Li XB (2017) The cotton beta-galactosyltransferase 1 (GalT1) that galactosylates arabinogalactan proteins participates in controlling fiber development. Plant J 89:957–971

Qu J, Ye J, Geng YF, Sun YW, Gao SQ, Zhang BP, Chen W, Chua NH (2012) Dissecting functions of KATANIN and WRINKLED1 in cotton fiber development by virus-induced gene silencing. Plant Physiol 160:738–748

Ramegowda V, Mysore KS, Senthil-Kumar M (2014) Virus-induced gene silencing is a versatile tool for unraveling the functional rel-evance of multiple abiotic-stress-responsive genes in crop plants. Front Plant Sci 5:323

Ryu CM, Anand A, Kang L, Mysore KS (2004) Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species. Plant J 40:322–331

Tuttle JR, Idris AM, Brown JK, Haigler CH, Robertson D (2008) Geminivirus-mediated gene silencing from Cotton leaf crumple virus is enhanced by low temperature in cotton. Plant Physiol 148:41–50

Tuttle JR, Haigler CH, Robertson D (2012) Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system. Plant Methods 8:27

Ullah A, Sun H, Yang X, Zhang X (2018) A novel cotton WRKY gene, GhWRKY6-like, improves salt tolerance by activating the ABA signaling pathway and scavenging of reactive oxygen species. Physiol Plant 162(4):439–454

Valentine T, Shaw J, Blok VC, Phillips MS, Oparka KJ, Lacomme C (2004) Efficient virus-induced gene silencing in roots using a modified tobacco rattle virus vector. Plant Physiol 136:3999–4009

Wang X, Tang C, Huang X, Li F, Chen X, Zhang G, Sun Y, Han D, Kang Z (2012) Wheat BAX inhibitor-1 contributes to wheat resist-ance to Puccinia striiformis. J Exp Bot 63:4571–4584

Wang M, Wang Q, Zhang B (2013) Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.). Gene 530:44–50

Watanabe N, Lam E (2008) BAX inhibitor-1 modulates endoplasmic reticulum stress-mediated programmed cell death in Arabidopsis. J Biol Chem 283:3200–3210

Watanabe N, Lam E (2009) Bax Inhibitor-1, a conserved cell death suppressor, is a key molecular switch downstream from a vari-ety of biotic and abiotic stress signals in plants. Int J Mol Sci 10:3149–3167

Wei Y, Xu Y, Lu P, Wang X, Li Z, Cai X, Zhou Z, Wang Y, Zhang Z, Lin Z, Liu F, Wang K (2017) Salt stress responsiveness of a wild cotton species (Gossypium klotzschianum) based on transcrip-tomic analysis. PLoS One 12:e0178313

Wu H, Tian Y, Wan Q, Fang L, Guan X, Chen J, Hu Y, Ye W, Zhang H, Guo W, Chen X, Zhang T (2018) Genetics and evolution of MIXTA genes regulating cotton lint fiber development. New Phy-tol 217:883–895

Xu Q, Reed JC (1998) Bax inhibitor-1, a mammalian apoptosis sup-pressor identified by functional screening in yeast. Mol Cell 1:337–346

You Q, Xu W, Zhang K, Zhang L, Yi X, Yao D, Wang C, Zhang X, Zhao X, Provart NJ, Li F, Su Z (2017) ccNET: database of co-expression networks with functional modules for diploid and poly-ploid Gossypium. Nucleic Acids Res 45:D1090-D1099

Zha H, Fisk HA, Yaffe MP, Mahajan N, Herman B, Reed JC (1996) Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells. Mol Cell Biol 16:6494–6508

Zhang J, Yu D, Zhang Y, Liu K, Xu K, Zhang F, Wang J, Tan G, Nie X, Ji Q, Zhao L, Li C (2017a) Vacuum and co-cultivation agro-infiltration of (germinated) seeds results in tobacco rattle virus

Plant Cell Reports

1 3

(TRV) mediated whole-plant virus-induced gene silencing (VIGS) in wheat and maize. Front Plant Sci 8:393

Zhang M, Han LB, Wang WY, Wu SJ, Jiao GL, Su L, Xia GX, Wang HY (2017b) Overexpression of GhFIM2 propels cotton fiber development by enhancing actin bundle formation. J Integr Plant Biol 59:531–534

Zhao J, Bai W, Zeng Q, Song S, Zhang M, Li X, Hou L, Xiao Y, Luo M, Li D, Luo X, Pei Y (2015a) Moderately enhancing cytokinin

level by down-regulation of GhCKX expression in cotton concur-rently increases fiber and seed yield. Mol Bred 35:60

Zhao J, Bai W, Zeng Q, Song S, Zhang M, Li X, Hou L, Xiao Y, Luo M, Li D, Luo X, Pei Y (2015b) Moderately enhancing cytokinin level by down-regulation of GhCKX expression in cotton concur-rently increases fiber and seed yield. Mol Breed 35:60

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