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GbAt11 gene cloned from Gossypium barbadense mediates resistance to Verticillium wilt in Gossypium hirsutum
Journal of Cotton Research volume 3, Article number: 9 (2020)
Gossypium hirsutum is highly susceptible to Verticillium wilt, and once infected Verticillium wilt, its yield is greatly reduced. But G. barbadense is highly resistant to Verticillium wilt. It is possible that transferring some disease-resistant genes from G. barbadense to G. hirsutum may contribute to G. hirsutum resistance to Verticillium wilt.
Here, we described a new gene in G. barbadense encoding AXMN Toxin Induced Protein-11, GbAt11, which is specifically induced by Verticillium dahliae in G. barbadense and enhances Verticillium wilt resistance in G. hirsutum. Overexpression in G. hirsutum not only significantly improves resistance to Verticillium wilt, but also increases the boll number per plant. Transcriptome analysis and real-time polymerase chain reaction showed that GbAt11 overexpression can simultaneously activate FLS2, BAK1 and other genes, which are involved in ETI and PTI pathways in G. hirsutum.
These data suggest that GbAt11 plays a very important role in resistance to Verticillium wilt in cotton. And it is significant for improving resistance to Verticillium wilt and breeding high-yield cotton cultivars.
Verticillium wilt is a highly destructive soil-borne fungal disease of plants that affects an extensive range of host species, including many agricultural crops (Burpee and Bloom 1978; Fradin et al. 2011; Qi et al. 2016). Verticillium wilt of cotton is caused by Verticillium dahliae, but how the pathogen causes the disease of plants remains to be clarified (Gao et al. 2011). However, there is an accumulative evidence to show that the toxin produced by V. dahliae is the main pathogenic factor, for example, a protein or glycoprotein is mainly responsible for the wilt symptoms. Studies on the resistance mechanisms of plants to Verticillium wilt found two genetic loci, Ve1 and Ve2 in tomato. The expression of both Ve1 and Ve2 in potato can enhance resistance to V. dahliae (Kawchuk et al. 2001; Simko et al. 2004). Although it has been shown that Ve1 gene is functional only in tomato (Fradin et al. 2011; Fradin et al. 2009), GbVe1, which is a homolog of the tomato Vel cloned from Gossypium barbadense, is highly resistant to Verticillium wilt, and its overexpression in Arabidopsis thaliana has confirmed the disease resistance function of this gene (Zhang et al. 2011; Zhang et al. 2013). Based on the genetic principle of immune response induction, we treated G. barbadense with purified V. dahliae filtrate protein, extracted RNA, and cloned 11 full-length cDNAs of specific genes (GbAt1 - GbAt11) using suppression subtractive hybridization (SSH). The objective of this study was to analyze the molecular role of GbAt11 gene in resistance of cotton to Verticillium wilt.
Recently, considerable progress has been made in understanding the immune mechanisms underlying the response of plants to pathogen infection. Studies have shown that plants identify pathogenic microorganisms by the presence of specific molecular motifs, known as pathogen-associated molecular patterns (PAMP)(Ozinsky et al. 2000), for example, the flagellin protein Flag22 on the surface of Pseudomonas syringae, the bacterial elongation factor EF-Tu, and chitin (Asai et al. 2002; Wan et al. 2008). When plants contact Flag22, the receptor protein FLS2 is activated on the surface of plant cells and is up-regulated through MAPK kinase signal transduction and transcription factor WRKY29 expression (Asai et al. 2002; Gomez-Gomez and Boller 2000; Adachi et al. 2015), therefore causing the production of reactive oxygen species (ROS) and the accumulation of callose at the infected site, thus protecting against the pathogen invasion (Nurnberger et al. 2004). FLS2-mediated disease resistance pathways are also involved in the BAK1 element in the brassinosteroids (BR) pathway, which is the co-receptor of both BR and FLS2 (Albrecht et al. 2008; Lin et al. 2013). After FLS2 is activated by Flag22, BAK1 is recruited to form a complex, and then transfers the resistance signal to downstream by a process called PAMP-Triggered-Immunity (PTI)(Albrecht et al. 2012). Some bacterial pathogens have evolved effectors to inhibit PTI; however, plants have evolved proteins to counteract these effectors, which can relieve the inhibition of effectors on PTI through a process called effector-triggered-immunity (ETI). Thus, host plants and their pathogens co-evolve during this mutually antagonistic process.
Since GbAt11 was induced in G. barbadense with high resistance to Verticillium wilt, we studied the function of GbAt11 in resistance to Verticillium wilt, and the GbAt11-G. hirsutum lines significantly increased resistance to Verticillium wilt. Similarly, we found that overexpression of GbAt11 can up-regulate the expression of FLS2, BAK1 and other related disease resistance genes. Therefore, we speculate that GbAt11 plays an important role in the process of resistance to Verticillium wilt and is beneficial for improving resistance to Verticillium wilt and breeding high-yield cotton cultivars. And it also has reference value for the study of the mechanism of plant antifungal diseases.
Materials and methods
G. barbadense variety H7124 seeds were purchased from the National Cotton Seeds Repository (China). Seeds of G. hirsutum variety JM169 were provided by the Institute of Cotton, Hebei Academy of Agriculture and Forestry Sciences (Shijiazhuang, Hebei). G. barbadense and G. hirsutum were grown in greenhouse at 25 °C with a 12 h/12 h (day/night) cycle, and G. hirsutum JM169 was used as the wild type (WT) control. All transgenic plants used in this study are in JM169 background.
GbAt11 gene cloning and sequencing
The G. barbadense seedlings were divided into two groups, and one was soaked in a solution of V. dahliae toxin, while the other was soaked in water as the control. Three plants were sampled at 6, 12, 18, and 24 h of treatment, respectively, while their total RNA was extracted, and mRNA was purified, then used as SSH (Diatchenko et al. 1996). A total of 11 specific gene fragments were obtained, cloned into T-vector and sequenced by Sangon Biotech Co. Ltd. From the sequencing results, they were named GbAt1 to GbAt11. This study focused on exploring the role of GbAt11 gene. Basic local alignment search tool (Blast) searches were performed against GenBank to find the full-length cDNA sequence. The chromosomal location of the GbAt11 gene was obtained from an analysis at the website.
Specific real-time PCR detection of GbAt11 expression in G. barbadense and G. hirsutum
Seedlings of G. barbadense and G. hirsutum were grown in a greenhouse. Leaves were collected at the adult-plant stage from the same parts of the plants, soaked in Vd991 spore suspension (1 × 106 per mL), sampled after 0 h, 4 h, 8 h, 12 h, and 24 h, respectively, and total RNA was extracted from 100 mg of powder. Then we used real-time polymerase chain reaction (RT-PCR) (Salin et al. 2005) to quantify the level of GhAt11-specific mRNA at different times. RNA was extracted with the EASYspin plant RNA rapid extraction kit (Beijing Aidlab biotechnologiesCo. Ltd), quantified, and the relative expression of GbAt11 mRNA was measured using RT-PCR after cDNA synthesis by reverse transcription. PCR primer sequences were designed as follows: forward primer 5′-AGGGTTTCGTCGTCTACT-3′, and reverse primer 5′-GATGCTTCTCCTCATAGG-3′. Amplification reactions (20 μL) contained 10 μL SYBR Premix Ex Taq, 0.6 μL of each primer (F + R), 0.4 μL ROX, 1–2 μL cDNA, and ddH2O to 20 μL.
Construction of the GbAt11 overexpression binary vector (the concentration of primary proteinsis quantified with spectrophotometer)
We replaced the HygR gene with the tfdA gene in the pCAMBIA1300 binary vector to make a vector called pSPT. We designed primers containing Sal1/KpnI restriction enzyme sites on both ends of the GbAt11 open reading frame (ORF); AT11–2-SalI-F: 5′-CGGTCGACATG TCGATCGCGTTGGAACG-3′, and AT11–2-KpnI-R: 5′-CGGGTACCGTTATATTCAC GTACATCAGCC-3′, and obtained the target gene by reverse transcription of G. barbadense AXMN mRNA. The pSPT vector was digested with SalI and KpnI, then ligated with the target gene fragment using T4 DNA ligase to construct the recombinant binary vector containing the GbAt11 gene.
Verticillium dahliae inoculations
V. dahliae inoculations was conducted according to Fradin etc. (2009). The 2~ 3-week-old cotton plants were uprooted, and then the roots and (or) adult-stage cotton leaves were rinsed in water. Subsequently, the roots or petioles were dipped for 24–72 h in a suspension of 106conidia per mL of Czapek’s medium dextrose broth (Difco) and harvested from 2-week-old Verticillium cultures on Czapek’s medium dextrose agar (Oxoid). Control plants were treated similarly, but their roots were dipped in Czapek’s medium without conidiospores. After dipping, the disease phenotype was recorded and photographed from 1 h to 72 h.
Screening of transgenic positive cotton lines
Transformation of G. hirsutum was performed using the pistil drip method (Zhang and Chen 2012). Transgenic seeds were sown in the field, sprayed with 2,4-D at 30 mg·kg− 1 after sprouting, and the plants with a completely unfolded true leaf were screened. DNA was extracted and plants were screened for the tfdA gene and the target gene. Primers for tfdA amplification were tfdA-F:5′-ATGAGATCCATGGGTGAGCG-3′, and tfdA-R:5′-AGAACGCAGCGGTTGTCC-3′. PCR was used to identify single copy transgenic plants based on the segregation ratio of the T2 generation against 2,4-D. We first identified disease resistance in the single copy lines in the disease nursery (cotton stalks inoculated with Vd991 were crushed and evenly spreaded in the field every year, to ensure the uniformity of V. dahliae in the disease nursery), and then we selected lines L-213, L-214, and L-235 with good field resistance, treated with a V. dahliae spore suspension (concentration of 1 × 106 per mL) for resistance identification in a disease assay. We next extracted total proteins from L-214 leaves, concentrated the GbAT11-Flag protein fusion using the Flag tag, fractionated the proteins on a denaturing polyacrylamide gel, and then detected the anti-Flag antibody by western hybridization.
Transcriptome analysis was performed by Beijing Biomarker Technologies Co. Ltd.
Subcellular localization of the GbAT11-GFP fusion protein
We constructed the pCAMBIA:GbAT11-GFP recombinant expression vector and used it to transform Arabidopsis thaliana. T2 generation transgenic seeds were sown on murashige and skoog basal medium (MS) medium, and after the GbAT11-GFP-expressing seedlings grew for 1 week, GFP fluoresence was observed with a laser scanning confocal microscope. The excitation and emitting wavelengths for GFP were 488 and 525 nm, respectively. This allowed us to see whether GbAT11 was localized to the cell wall after treated with V. dahliae toxin. We treated the roots of transgenic Arabidopsis with 20% sucrose solution to plasmolyze the cells in order to observe the membrane localization of GbAT11.
Field disease resistance identification and examination of yield traits
The test on Verticillium wilt resistance of plants carrying the endogenous At11 gene, transgenic plants overexpressing GbAt11, and G. hirsutum carrying a TALENT knockout of GhAt11 was carried out in a field disease nursery inoculated with the Vd991 in Shandong province. There were two lines of each variety, a total of 60 plants. Plants were examined for symptoms of Verticillium wilt on June 20 and August 20, and were classified into five levels based on the degree of morbidity (ratings of 0 to 4). We calculated the Verticillium wilt disease index according to the formula (1), for every line, and counted the average number of bolls per plant on September 10. And the same test was carried out at the test base of China Agricultural University (Hebei province, in north-central China).
Screening of Gossypium barbadense-response genes induced by Verticillium wilt using suppressive subtraction hybridization
Gossypium barbadense possesses the highest level of resistance to verticillium wilt among the four cotton species, while the widely cultivated G. hirsutum is highly susceptible. We soaked seedlings of G. barbadense and G. hirsutum in a solution of purified proteins secreted by V. dahliae, and necrotic lesions were found present on the leaves of G. barbadense after 72 h, while the entire leaves of G. hirsutum were withered (data not shown). We induced the verticillium wilt-resistant G. barbadense with a solution of purified V. dahliae proteins, and obtained 11 specific genes (GbAt1-GbAt11) using SSH.
We initially focused on GbAt11 because its expression appeared to be strongest in G. barbadense induced by V. dahliae. The full-length GbAt11 cDNA is 768 bp, and the predicted molecular mass of the protein is about 27 kd. The N-terminal ORF from G. barbadense has an additional 17 amino acids than the predicted protein from G. hirsutum. In addition, BLAST searches confirmed that no GbAt11-like gene was present in A. thaliana. Further DNA sequence comparison and amino acid sequence analysis predicted that GbAT11 has 49.08% and 48.76% amino acid sequence homology with MTD1 (Methylene-tetrahydrofolate dehydrogenase [NAD(+)]) and Jatropha curcas serine/arginine repetitive matrix protein 2-like (SRRM2-like), respectively. But the strange thing is that the primary amino acid sequence of GbAT11 is closely similar to Damaged DNA-binding 2, putative isoform 1 (DDB2), but the gene has not yet been reported, and the most similar homolog gene is related with the uncharacterized protein LOC105800652 in NCBI (National Center for Biotechnology Information). A further search of the COTTONGEN (http://www.cottondb.org) indicated that the GbAt11 gene is located on Chromosome 9 in G. raimondii, so GbAt11 is a new gene which may be located on chromosome 9 in G. hirsutum.
GbAt11 gene expression is specifically induced by Verticillium dahliae in Gossypium barbadense
In order to investigate the relationship between GbAt11 and V. dahliae, and to explore transcriptional difference between G. barbadense and G. hirsutum, we collected young leaves at the adult-plant stage from the same parts of wild G. barbadense and wild G. hirsutum. Leaves were dipped in filtration of Vd991 (spore concentration of 1 × 106per mL). Total RNA was extracted from samples at different induction times, then reversely transcribed into cDNA to determine the relative level of GbAt11 transcription by RT-PCR. Results showed that: (1) transcription of GbAt11 increased 10-fold in seedlings treated with Vd991 for 4 h in G. barbadense, and then declined to the initial level. Transcription of GbAt11 increased significantly when induced for 8 h, and peaked at 24 h at more than 70-fold. These results indicated that the GbAt11 gene is specifically induced by V. dahliae in G. barbadense. (2) Transcription of GhAt11 increased slightly in G. hirsutum, but the leaves were heavily wilted at 12 h, and RNA were failed to be extrated. These results showed that expression of GbAt11 is specifically induced by V. dahliae, and is closely related to the host resistance response to pathogenic bacteria. In response to inducing by V. dahliae, GbAt11 is specifically overexpressed in Verticillium wilt-resistant G. barbadense, but the homolog of this gene was not specifically induced in G. hirsutum which is susceptible to Verticillium wilt, and its leaves appeared serious wilting within 12 h (Fig. 1).
GbAt11 overexpression enhances resistance to Verticillium wilt in Gossypium hirsutum
Because GbAt11 is specifically induced in highly Verticillium wilt-resistant G. barbadense, we speculated that overexpression of GbAt11 could improve the resistance to Verticillium wilt in susceptible G. hirsutum. To test this hypothesis, we transformed the wilt-susceptible G. hirsutum variety JM169 (WT) with GbAt11 and obtained 52 transgenic plants. Verticillium wilt-resistance ratings of field and greenhouse in a disease nursery were performed on the T2 generation plants of 17 transgenic lines. L-214 is a high-yielding line with good disease resistance. As shown in Fig. 2a, compared with the parental line (WT), L-214 showed the highest disease resistance, followed by L-213, but L-235 showed poor disease resistance.
In order to further confirm the role of GbAt11 gene in disease resistance, we extracted total proteins from leaves of transgenic L-214 (the flag tag fused to the C-terminal end of GbAT11) and the parental line WT (No flag tag). Anti-flag antibody-coated magnetic beads were incubated with the total protein to capture the flag tagged GbAT11 (IP). Then we used anti-flag antibody for Western blot detection, which showed that transgenic L-214 had one hybridizing band between 25 and 35 kd, which was absent in JM169, confirming the presence of GbAT11 protein in L-214 (Fig. 2b). To shed light on the relationship between the level of GbAt11 transcription and disease resistance, we quantified GbAt11 expression in transgenic lines L-213, L-214, and L-235 using real-time PCR. The results showed that GbAt11 expression was the highest in L-214 with the best Verticillium wilt resistance, and successively decreased in lines L-213 and L-235 (Fig. 2c), corresponding to the relative levels of Verticillium wilt resistance in the two lines (Fig. 2c and d). In subsequent experiments, L-214 was selected as its excellent disease resistance.
The disease index was calculated as formula (1):
Here, disease rating c was scored in five grades (0 to 4) based on the severity of Verticillium wilt symptoms in cotton. Grade 0, health, no disease symptom of leaves; grade 1, leaves have slight disease symptoms; grade 2, leaves have disease symptoms in moderate degree; grade 3, leaves have remarkable disease symptoms; And grade 4, leaves have severe disease symptoms. Plant number c was the number of plants corresponding to the rating of disease severity. The '4' was grade 4 which was the highest grade.
GbAt11 overexpression in Gossypium hirsutum up-regulates transcription of several key genes in the immunity pathways
Given that GbAt11 overexpression can improve disease resistance in plants, further transcriptome analyses (Wang et al. 2009) were performed in L-214 line and WT line. Clustering differentially expressed genes showed that gene transcription was significantly altered in the transgenic GbAt11-expressing line compared with the parent line. A total of 472 genes showed significant expression differences (FDR < 0.01)(Fig. 3a). Further transcriptome analysis showed that many genes involved in “plant-pathogen interactions” (KEGG Pathway) were up-regulated in transgenic plants of GbAt11, including FLS2 and the calcium dependent protein kinase CDPK (Harmon et al. 1994; Rigo et al. 2013); RPS2, RIN4 (Rigo et al. 2013) and PBS1 (Joys 1965), which are disease-resistance genes in ETI; and HSP90, the downstream gene in ETI. Therefore, it is reasonable to speculate that the transgenic GbAt11 line can activate ETI by affecting post-translational modifications, protein transporting and folding, relieving the inhibition of the pathogen on PTI-based immunity and improving resistance to Verticillium wilt in cotton. To confirm the result of transcriptome, real-time PCR was conducted in three lines, and the data showed that FLS2 in L214 and L213 is extremely significant higher than WT (Fig. 3b). Nextly, 7 genes involved in ETI and PTI process were assayed via real-time PCR in L214. The results confirmed that FLS2, BAK1, MKK2, FRK1, RIN4, RPS2 genes were significantly up-regulated in transgenic line L-214, and the FLS2 gene showed the highest up-regulation (Fig. 3c).
Subcellular localization of GbAT11 in Arabidopsis thaliana
We used Arabidopsis thaliana to explore the functional site of GbAT11 at the cellular level. We first produced transgenic plants of Arabidopsis thaliana expressing GbAt11-gfp, and then observed the subcellular localization of the GbAT11-GFP fusion protein in the root tip by laser scanning confocal microscopy. The results showed that GbAT11 is mainly localized to the nucleus (Fig. 4a), and a small amount presents on the cell wall (Fig. 4b). To clarify whether GbAT11 is also located on the cell membrane in addition to the nucleus, we treated Arabidopsis root tips with a 20% sucrose solution to separate cytoplasm from the cell wall, and then observed them under a fluorescence microscope. We found that the GbAT11-GFP protein was indeed located on the cell membrane (Fig. 4c). We also observed that the fluorescence intensity of the cell membrane was enhanced in Fig. 4c, which could be caused by shrinkage of the cell membrane, or the transfer of the GbAT11-GFP fusion protein from the nucleus to the membrane under osmotic stress.
GbAt11 overexpression increase cotton yield
A common misconception is that it is difficult to have both disease resistance and high yield in the same plant. However, our results showed that overexpression of GbAt11 not only improved disease resistance, but also increased boll-setting, which is the most important factor in cotton production. To identify the high yielding transgenic lines that overexpress GbAt11, we investigated disease severity of the transgenic lines in a V. dahliae nursery and also analyzed the basic yield traits, such as boll number per plant, boll opening phenotype in the field (Fig. 5a). The disease indexes of Verticillium wilt were 6.52, 7.35, and 9.09 in L-214, L-213, and L-235, respectively, and 69.44, 60.23, 6.25, and 61.36 in the wild type control (WT), the susceptible control (SCK), the disease resistant control (RCK), and the high yield control (YCK), respectively. Through analysis, it was found that the disease indexes of all transgenic lines were lower than the WT, YCK, and SCK, moreover, L-214 was extremely close to the RCK (Fig. 5b). The results showed that the bolls per plant of L-214, L-213 and L-235 were 14.9, 17.6 and 20.0, respectively, and 12.5, 8.9, 11.2, and 11.7 in WT, SCK, RCK, and YCK, respectively. Boll-setting in all three transgenic lines was higher than the parent lines and other controls. Other than these three lines, the bolls per plant in many other different lines were also better than YCK (Fig. 5c). In conclusion, overexpression of the GbAt11 gene in G. hirsutum not only improved its resistance to disease caused by V. dahliae, but also increased the number of bolls per plant in a field that was severely infested with Verticillium wilt.
In this study, GbAt11 gene expression experiments showed that GbAt11 up-regulation started at 4 h after induction in G. barbadense, then decreased at 8 h, and increased to 70-fold at 24 h. This result is a normal response of plants to pathogen infection: the expression of genes associated with disease resistance will increase, followed by a process of adaptation, and then expression increases again as the infection becomes severe (Denance et al. 2013). This pattern of GbAt11 expression was observed in the response of G. barbadense to infection by V. dahliae. As for the highly susceptible G. hirsutum, expression of genes relevant to disease resistance are not induced, but occurs systemic infection and results in leaf wilt. Thus it appears that GbAt11 plays an important role in determining Verticillium wilt resistance in G. barbadense.
Previous studies have shown that after low temperature treatment, dehydrated protein WZY2 will leave the dehydration point and disperse into the cytoplasm, indicating that low temperature will affect the distribution of wheat dehydrated protein WZY2 in cells (Lv 2014). Observing the localization of GbAT11, we can find that the cell membrane leaves the cytoplasm after the plasma wall separation occurs, and the fluorescence is localized in the cell membrane.The fluorescence brightness of the cell membrane in Fig. 4c is enhanced, which may be caused by shrinkage of the cell membrane, or when the cell is stressed, the GbAt11-GFP fusion protein localized in the nucleus is transferred to the cell membrane.
Typically, overexpression of transcription factors, such as DRE, can improve stress tolerance or disease resistance in plants, but it may also cause dwarfing and yield reduction, etc. (Narusaka et al. 2003). We found that the cotton strain transferred to GbAt11 had a significant effect against Verticillium wilt. In addition, later production of cotton showed that the yield of transgenic cotton was higher than wild type. Although we demonstrated that the transgenic lines overexpressing GbAt11 had better resistance to Verticillium wilt, the mutant strain was failed to be constructed. Because cotton is a heterotetraploid plant, and AT11 has two copies in G. barbadense, it is difficult to knock them out together with the same vector. In addition, because of the limitation of cotton growth cycle, it is impossible to obtain double mutant in a short time. So next we will use the virus induced gene silencing system to silence the GbAt11 gene, and then further investigate the cotton resistance to Verticillium wilt. Unfortunately, we were unable to identify any proteins that directly interact with GbAT11 by co-immunoprecipitation and yeast two hybrid assays, so this remains to be further investigated in the future. Although its function needs to be further researched, the effect of increasing yield and resistance to Verticillium wilt has guiding significance to cotton production practice.
GbAt11 has been identified as a new gene which is specifically induced by V. dahliae. Its overexpression in G. hirsutum not only significantly improves resistance to Verticillium wilt, but also up-regulates transcription of several key genes in the immunity pathways. These results show that GbAt11 plays a role in disease resistance. Furthermore, overexpression of the GbAt11 gene in G. hirsutum can also increase the number of bolls per plant to increase cotton yield.
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We thank Prof. GONG Zhizhong and Prof. LI Jigang of the Department of Plant Science, College of Biological Sciences of China Agricultural University for their assistance. We gratefully acknowledge the State Key Laboratory of Plant Physiology and Biochemistry for allowing this work to take places in their laboratory.
This research was financially supported by the National Key Research and Development Program of China (2016YFD0101904).
This study was approved by the local ethics committee.
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The authors declare that they have no competing interests.
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QIU, T., WANG, Y., JIANG, J. et al. GbAt11 gene cloned from Gossypium barbadense mediates resistance to Verticillium wilt in Gossypium hirsutum. J Cotton Res 3, 9 (2020). https://doi.org/10.1186/s42397-020-00047-3
- Gossypium barbadense, GbAt11, Verticillium wilt, resistance