Identification and bioinformatics analysis of BONZAI in cotton
Based on the BONZAI contributed to disease resistance in Arabidopsis, it is very also essential to elucidate the roles of the BONZAI in cotton. Since the loss of AtBON1 function in Arabidopsis results in increasing disease resistance in response to pathogen (Yang and Hua 2004), we speculate that its homologs in cotton might mediate resistance as well to Verticillium wilt. First of all, the cDNA sequences of AtBON1, AtBON2 and AtBON3 blasted search the genome sequences of cotton database to identify BONZAI genes in cotton. Seven cDNAs (Sequence ID: Gh_A03G0485, Gh_D03G1053, Gh_D09G2399, Gh_D11G0417, Gh_A11G3017,Gh_A13G0813 and Gh_D13G1054) were identified and further named as GhBON1, GhBON2 and GhBON3 to show the type I and type III BONZAI they each belong to. The deduced GhBON1, GhBON2 and GhBON3 protein sequences have almost 100% sequence identity to AtBON1, AtBON2 and AtBON3. Because the cotton is allotetraploid and has A and D kinds of genomes, three cDNA sequences corresponding to the GhBON1, two genes corresponding to GhBON2 and two genes corresponding to GhBON3 were found in cotton genome sequences which showed the complex of the cotton genomes. Phylogenetic tree analysis the BONZAI proteins in Arabidopsis and cotton revealed that Gh_A03G0485, Gh_D03G1053 and Gh_D09G2399 are closely related to the AtBON1 protein, Gh_D11G0417 and Gh_A11G3017 are closely related to AtBON2 protein and Gh_A13G0813 and Gh_D13G1054 are closely related to AtBON3 protein (Fig. 1a). According to the previous classification of plant BONZAI genes (Zou et al. 2016), Gh_A03G0485, Gh_D03G1053, Gh_D09G2399, Gh_D11G0417 and Gh_A11G3017 belong to type I while Gh_A13G0813 and Gh_D13G1054 belong to type III BONZAI. We further checked the structure of the homeologous copies of GhBON1, GhBON2 and GhBON3 genes from upland genome sequences. All these prutative GhBONs proteins contain two C2 domains (C2A and C2B) at the N-terminus and a vWA domain at C-terminus with the features of BONZAI (Fig. 1b). The homeologous copies of GhBON1, GhBON2 and GhBON3 have almost similar sequences in amino acid sequences of these domains. Thus, there are inferred that the cotton has three homeologous GhBON1 genes, two homeologous GhBON2 genes and two homeologous GhBON3 genes.
GhBON1, GhBON2 and GhBON3 response to V. dahliae
In order to determine GhBON1, GhBON2 and GhBON3 against V. dahliae, the expression levels of GhBON1, GhBON2 and GhBON3 against V. dahliae was performed by qRT-PCR. Total RNA was isolated from inoculated by V. dahliae isolate V991 treatment. Specific primers were designed to distinguish between GhBON1, GhBON2 and GhBON3 but they were not able to distinguish the corresponding homeologous copies. As shown in Fig. 1c, GhBON1, GhBON2 and GhBON3 expression were all obviously induced by V. dahliae, which further confirms the finding that GhBONs may be involved in V. dahliae response.
Furthermore, the expression of GhBON1 was also used to investigate the response to various abiotic stresses including 200 mM methyl jasmonate, 2 mM SA, and 1 mM H2O2 stresses. These results supported the view that expression of GhBON1 was positively induced by SA, JA and H2O2. Especially, it could be inducible obviously by JA and H2O2 treaments (Fig. 1d). Therefore, it may come to a conclusion that GhBON1 expression obviously responds to V. dahliae which mediated by jasmonic acid and ROS pathways in cotton.
Reduced V. dahliae sensitivity in GhBONs-silencing cotton plants
To ascertain whether GhBONs functions in responds to V. dahliae, we employed the tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) technology to silence the expression of GhBONs in cotton. Because there are three homeologous GhBON1, two homeologous GhBON2 and GhBON3 in the upland cotton genome, we constructed respectively the approximately 300 bp GhBONs CDS which was integrated into the vector pTRV2 (TRV: GhBON1, GhBON2 and GhBON3) to suppress GhBONs. When the fresh true leaves which infected by the pTRV2 vector with a fragment of GhCLA1 (cloroplastos alterados1) appeared albino phenotype or TRV:00 vector without DNA insertion, the VIGS efficiency indicator or a control, indicated that the VIGS system worked efficiently under the experimental conditions (Fig. 2a), and then the fresh leaves and roots from TRV:00 and TRV: GhBON1, TRV: GhBON2 or TRV: GhBON3 plants were harvested. All the expression of TRV:GhBONs was dramatically reduced in VIGS plants with the qRT-PCR analyses (Fig. 2b). At the time was the three-leaf stage of the cotton plants, and then the plants were inoculated with V991. After about 16 days the growth of GhBON1, GhBON2 and GhBON3-silenced plants through phenotypic analysis showed that their resistance toV991 was enhanced compared to that of TRV:00 plants. The leaves of TRV:00 plants displayed more serious wilting and chlorosis symptoms than those of TRV: GhBONs (Fig. 2c), the disease rate of TRV: GhBON1, TRV: GhBON2 and TRV: GhBON3 plants were obviously higher than TRV: 00 plants (Fig. 2d), the disease index also clearly decreased than TRV: 00 plants, respectively (Fig. 2e). Moreover, the brown changes of vascular bundles of GhBONs-silenced plants were more obvious difference from that of corresponding control plants and had significantly brown spots than the control (Fig. 2f). We further measured fungal recovery in stem segments of infected cotton plants, the TRV:GhBONs plants fungi colonized were slower than the TRV: 00 plants (Fig. 2g). These results suggest that the silencing of GhBONs promotes the resistance of cotton to V. dahliae, which GhBONs negativly regulates the resistance of plants to pathogen infection.
Silencing of GhBONs activated ROS mediated plant disease-resistance response signaling pathways
Given that GhBONs function as a negative regulator against V. dahliae infection and could also be induced by H2O2 stress, we speculate that the increased resistance to V. dahliae infection in GhBONs-silenced plants may lead to the changes of the redox status. To determine this possibility, The production of total ROS was examined in the leaves of GhBONs-silenced and control plants with V. dahliae infection. GhBONs-silenced leaves after V. dahliae infection were observed to accumulate more ROS than the control with DAB (3,3’-diaminobenzidine tetrahydrochloride) staining (Fig. 3a). This finding indicated that silencing of GhBONs-silenced leaves increased cell death in the infected leaves and also confirmed that GhBONs function in ROS burst that occurs challenging V. dahliae attack, thereby inducing more cell death.
To determine whether GhBONs knock-down has other effect on V. dahliae infection in silencing plants, we further select several genes to monitor their expression patterns such as PR1, PR4, NPR1 and PDF1.2 mediated pathogen stress-responsive. The transcript of these genes was induced in GhBONs-silencing plants and enhanced to a high level under V. dahliae infection conditions (Fig. 3b). These results were confirmed that GhBONs might participate in relative signaling pathways and further regulate pathogen stress-responsive than those in the control plants
GhBON1 interacts with GhBIR1 or GhBAK1, and the complex localizes at plasma membrane
To elucidate the regulatory function of GhBON1 in responds to disease resistance in cotton, full-length GhBON1 was used to identify potential interacting proteins using a yeast two-hybrid (Y2H) library of cotton. Several candidate GhBON1-interacting proteins were found, among which GhBIR1and GhBAK1 could interacts with GhBON1 and then we focused on the two genes. Firstly, a sets of cDNA constructs including the full-length, or truncated versions of GhBIR1 were generated to be sure which domain of GhBIR1 interacts with GhBON1. The full-length GhBIR1 (BIR1-FL), the entire intracellular domain of GhBIR1 (BIR1-JKC), the intracellular domain with both the C-terminal and JM domains deleted (BIR1-KD), and the intracellular C-terminal domain (BIR1-CT) were constructed (Fig. 4a) and transformed into the expressed GhBON1 yeast cells. It was found that BIR1-JKC could interact with BON1 while BIR1-FL, BIR1-KD and BIR1-CT were all unable to interact with BON1 in the yeast two-hybrid assay (Fig. 4b). These results demonstrate that the kinase domain of GhBIR1 is essential for the interaction between GhBIR1 and GhBON1 which is consistent with previously reported (Wang et al. 2011).
We next further tested the interactions of GhBIR1 and GhBON1 protein using a bimolecular fluorescence complementation (BiFC) assay in N. benthamiana leaves (Walter et al. 2004). GhBON1 was fused to an N-terminal yellow fluorescent protein (YFPN), and GhBIR1 was fused to a C-terminal YFP (YFPC). When GhBON1-YFPN was co-expressed with BIR1-YFPC in N. benthamiana leaves, a bright fluorescence signal was observed at the plasma membrane (Fig. 4c). Either the co-expression of BON1-YFPN and unfused YFPC or the co-expression of BIR1-YFPC and unfused YFPN could not produce any detectable fluorescence. Meanwhile, GhBIR1 expression was obviously induced by V. dahliae (Fig. 4d). These results suggest that GhBIR1 and GhBON1 interact with each other at the plasma membrane, which confirms that the complex may be involved in V. dahliae response.
Due to AtBON1 interacts physically with the AtBAK1 protein (Wang, et al. 2011), it was interesting to determine whether GhBON1 could also associate with GhBAK1. Similarly, the interaction of GhBAK1 with GhBON1was found in the yeast two-hybrid assay (Fig. 5a). Followed in the BiFC experiments in N. benthamiana leaves, GhBON1 was fused to YFPN and GhBAK1 was fused to YFPC. When GhBON1-YFPN and GhBAK1-YFPC was co-expressed in N. benthamiana leaves, a YFP signal was also observed at the plasma membrane (Fig. 5b), indicating a close interaction between GhBON1 and GhBAK1 in plants. Furthermore, GhBAK1 expression was affected and induced obviously by the pathogen V. dahliae infection (Fig. 5c).
Taken together, these results pointed out that there is closely association among GhBON1, GhBIR1 and GhBAK1 in response to V. dahliae.