1. Introduction
Cassava (
Manihot esculenta Crantz), known as the “king of starch”, is the fourth most significant source of carbohydrates for human consumption in tropical regions, behind rice, sugar, and maize [
1]. This versatile crop is a vital provider of sustenance and economic livelihood, supporting approximately 1 billion people worldwide. However, the threat posed by cassava bacterial blight (CBB), is caused by
Xanthomonas phaseoli pv.
manihotis (
Xpm), poses a significant challenge in tropical regions, where cassava cultivation is widespread [
2,
3]. CBB is a devastating foliar and vascular disease that can lead to substantial yield losses, severely impacting both the economic viability of cassava farming and food security in these areas [
4]. The management of CBB predominantly relies on the resistance inherent in the host plant. However, the emergence of new strains of
Xpm that can overcome this resistance calls for continuous research and development efforts to identify and cultivate cassava varieties that are resistant to these strains [
5]. In extreme cases, it can cause a significant decrease in cassava production, ranging from 50 to 75%, potentially leading to famine [
6]. Consequently, enhancing the natural resistance of cassava to CBB is crucial to mitigate yield losses. Notably, Zhu et al. successfully isolated the strain
Xanthomonas phaseoli pv.
manihotis strain CHN01 (
Xpm CHN01) using PacBio single-molecule real-time (SMRT) sequencing technology. This pathogen was found to have a genome consisting of a roughly 4.79 Mb circular chromosome and four plasmids. This discovery opens up new avenues for developing prevention and control strategies against CBB [
7].
Since plant disease resistance requires the coordination of multiple defense response genes, transcription factors (TFs), as key regulatory factors of gene expression, play crucial roles in plant disease resistance processes. There are reports dating back to early times indicating that basic helix–loop–helix (bHLH) transcription factors can interact with resistance genes to regulate the synthesis of compounds such as flavonoids. As early as 1989, Lc, a product of the
R gene, was the first plant protein reported to have a bHLH domain, involved in controlling the biosynthesis of flavonoids and anthocyanins in maize [
8]. The bHLH transcription factors consist of two α-helices that mediate dimerization and a basic domain that binds to E-box DNA sequences [
9]. bHLHs are important in promoting plant tolerance or adaptation to adverse environmental conditions [
10]. bHLHs often cooperate with members of other transcription factor families to control the regulation and induction of the biosynthesis of various secondary metabolites, mainly including terpenes and anthocyanins, which play crucial roles in mediating the interaction between plants and their environment [
11,
12].
Additionally, it has been reported that many bHLH transcription factors, such as those involved in abscisic acid (ABA) and auxin signal transduction, participate in the regulation of various hormone signaling pathways, and many have been shown to participate in the jasmonic acid (JA) signaling pathway [
13]. Cao et al. identified a tomato bHLH transcription factor,
SlJIG, as a MeJA-inducible gene, which exhibits strong induction upon MeJA treatment. Knocking out
SlJIG also leads to reduced expression of JA-responsive defense genes, indicating that
SlJIG is a direct target of MYC2, forming the MYC2-
SlJIG module, plays a role in terpene biosynthesis and resistance against cotton bollworm and
Botrytis cinerea [
14]. Yoodee et al. showed that pre-treatment with salicylic acid (SA) or methyl jasmonate (MeJA) 24 h before
Xpm inoculation enhanced the defense responses of two cassava cultivars. The severity of the disease decreased by 10% in the SA/MeJA-induced resistant cultivar (HB60) and significantly by 21% in the susceptible cultivar (HN) [
15]. Cao et al. characterized the biological function of Crf1, a bHLH transcription factor, in the development and pathogenicity of
P. oryzae through functional genetics, molecular, and biochemical methods [
16]. Members of the bHLH transcription factor family, including MYC3 and MYC4 from the same family as MYC2, interact with JAZ proteins, contributing to important functions such as growth inhibition, promotion of leaf senescence, and defense against diseases and pests [
17,
18,
19,
20]. Susceptibility of
TabHLH060-overexpressing
Arabidopsis plants to
Pseudomonas syringae DC3000 were significantly enhanced [
21]. Transgenic rice plants overexpressing
OsbHLH034 exhibited a JA-hypersensitive phenotype and increased resistance against rice bacterial blight [
22].
However, the role of bHLH transcription factor family in CBB resistance in cassava is not fully understood. The is study aimed to analyze the expression pattern of the MebHLH149 gene and its function in cassava response to CBB. This will provide a new perspective for future molecular breeding of disease-resistant cassava.
3. Discussion
Over the last 10 years, CBB has significantly affected the yield of cassava, a critical staple crop. Despite its importance, our understanding of resistance genes associated with this disease is still limited. The present study was designed to investigate the expression patterns of the MebHLH149 gene in cassava when exposed to Xpm CHN01 infection and hormone stress, to shed light on its potential role in the plant’s immune system. The findings indicate that MebHLH149 is actively involved in the early stages of the plant-pathogen interaction during Xpm CHN01 infection, implying a role in the defense mechanism of cassava. Additionally, this study identified MebHLH149 as an elements responsive to key plant hormones, namely MeJA, SA, and ABA. To further understand the potential signaling pathways influenced by MebHLH149, the expression patterns of genes associated with the ABA, MeJA, and SA pathways were examined. The data revealed a notable increase in the expression of genes related to these hormone pathways follow foliar application of the respective hormones. This observation supports the hypothesis that MebHLH149 plays a role in the signaling cascades mediated by ABA, MeJA, and SA.
Plant hormones are essential in orchestrating the immune responses of plants, creating complex networks that help combat a variety of diseases caused by external factors. These hormones form a protective barrier that not only prevents the progression of disease but bolsters the plant’s overall defense capabilities [
24,
25]. The significance of SA in plant disease resistance was first reported by White et al., who found that the application of SA to tobacco plants induced symptoms of tobacco mosaic virus (TMV). Intriguingly, these plants also displayed reduced lesion size, with some showing up to a 90% decrease post-infection, highlighting the ability of SA to enhance the resistance of tobacco to TMV [
26]. This discovery underscores the importance of this study in clarifying the novel role of the
MebHLH149 gene in the immune response of cassava when treated with plant hormones.
To address the reduction in cassava yield caused by
Xpm, previous studies employed VIGS as a means to validate the disease-resistance capabilities of various genes. This VIGS technology effectively silences specific target genes within plants, eliminating the need to create transgenic lines or conduct extensive mutant screenings. Its efficiency and simplicity make it particularly advantageous for exploring genes that are involved in plant growth, defense mechanisms, and other critical functions, rendering it an ideal tool for gene function discovery in plant seedlings [
27]. For example, prior investigations utilized VIGS to silence the
MeDELLA1,
MeDELLA2,
MeDELLA3, and
MeDELLA4 genes in cassava, aiming to understand their roles. Plants with these genes silenced showed reduced expression levels of genes associated with defense, suggesting a weakened ability to fend off CBB [
28]. In a similar vein, a different study scrutinized the expression patterns of three
MeWHYs genes under various conditions and then applied VIGS to silence them. The silencing of these genes resulted in an upsurge in bacterial populations and a concurrent decline in the disease resistance of cassava [
29]. Following this precedent, we applied VIGS to investigate the role of the
MebHLH149 gene in disease resistance. Our findings revealed that once the
MebHLH149 gene was successfully silenced, subsequent inoculation with
Xpm CHN01 led to more extensive water-soaked lesions and a greater bacteria count when compared to the negative control group. This outcome underscores diminished resistance in cassava following the silencing of the
MebHLH149 gene, thereby highlighting its positive regulatory influence in the plant’s defense mechanisms against CBB.
The resistance of cassava to the CBB pathogen is significantly enhanced by the
MebHLH149 gene, which may function through its combined action with its regulated genes. However, the scarcity of resistance gene germplasm, high genetic heterozygosity, heterozygous polyploidy, and low fertility in cassava present challenges for conventional breeding methods aimed at crop improvement. Given these challenges, progress in conventional breeding is often lengthy and arduous. Consequently, genetic engineering has become an invaluable alternative and complementary approach for enhancing cassava desirable agronomic traits [
30]. In the context of this study, we constructed a plant overexpression vector and successfully obtained transgenic seedlings. The ultimate aim was to investigate whether the
MebHLH149 gene confers enhanced resistance to CBB in the overexpressing cassava plants.
This study used the STRING database to predict potential interaction between the MebHLH149 protein and two candidate proteins, MePRE5 and MePRE6. We further explored their respective expression patterns to gain insights into the involvement of these proteins during the cassava–Xpm interaction. The findings revealed that the expression levels of both MePRE5 and MePRE6 genes were negatively correlated with the silencing of the MebHLH149 gene; conversely, their expression was positively influenced by the overexpression of MebHLH149. This correlation implied potential interaction between MebHLH149 with the MePRE5 and MePRE6 proteins. To confirm these interactions, we employed two complementary experimental techniques, Y2H and BiFC assays. These results highlight the biological significance of the MebHLH149 interaction with MePRE5 and MePRE6.
In conclusion, this study established that the MebHLH149 protein plays a pivotal role in three key plant signaling pathways: ABA, MeJA, and SA. These pathways are indispensable to the plant’s defense mechanisms against various biotic stressors. Notably, MebHLH149 exerts a positive regulatory impact on the cassava’s resistance to CBB, a devastating condition that can significantly impact crop yields and quality. By enhancing the plant’s ability to respond to pathogens through its involvement in these critical signaling cascades, MebHLH149 contributes to strengthening the plant’s overall disease resistance and resilience.
4. Materials and Methods
4.1. Plant Material and Strains
The cassava varieties South China No. 9 (SC9) and ZME642 are cultivated in the National Cassava Germplasm Repository. Situated in Danzhou, Hainan Province, China, with geographical coordinates of 109°30′ E and 19°30′ N, the repository offers an ideal environment for the cultivation and research of this specific cassava variety.
Nicotiana benthamiana plants were cultivated under controlled environmental conditions within a temperature-controlled chamber at the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences. The complete genomic data of Chinese
Xpm strain CHN01 have been deposited to the NCBI GenBank database under accession nos. CP083575, CP083576 (pXPM38), CP083577 (pXPM44), CP083578 (pXPM45), and CP083579 (pXPM48) [
7]. This strain was kindly provided by the Key Laboratory of Tropical Biological Resources Sustainable Utilization, Hainan University, Hainan Province, China.
4.2. Quantitative Real-Time PCR
Total RNA extraction and reverse transcription experiments from leaves were performed using the RNAprep Pure Plant Plus Kit (TIANGEN, Beijing, China) and the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) under the manufacturer’s instructions. The reverse-transcribed cDNA template was diluted tenfold for utilization as the template for quantitative real-time PCR and mixed with 2× ChamQ Universal SYBR qPCR Master Mix* (Vazyme, Nanjing, China) and primers for real-time fluorescence quantitative PCR analysis (ABI/QuantStudio 6 Flex, Singapore). Each experiment was carried out with at least 3 biological replicates, and data analysis was executed using the 2
−ΔΔCt method. Data analysis employed Dunnett’s test as the post hoc test following ANOVA (analysis of variance). Primer sequences are presented in
Table S1.
4.3. Xpm CHN01 and Hormone Stress in Cassava
To investigate the function of the
MebHLH149 gene in the immune response of cassava, leaf samples of the cassava variety SC9 were inoculated with
Xpm CHN01 and samples were collected at 0 h, 1 h, 3 h, and 6 h, followed by the analysis of
MebHLH149 gene expression patterns using RT-qPCR (using 0 h as the control) [
31]. To induce hormonal stress in cassava, ABA, MeJA, and SA were sprayed onto the leaves, samples were collected at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h to investigate the expression patterns of
MebHLH149 and related genes (using 0 h as the control) [
32]. The concentrations of the hormones utilized in these experiments were 100 μmol/L MeJA, 100 μmol/L ABA, and 2 mmol/L SA. Primer sequences employed in the present study are provided in
Table S1.
4.4. Identification of MebHLH149
The accession number of the cassava
bHLH149 gene is Manes.09G177600. The coding region (CDS) sequence of the cassava
bHLH149 gene, which is 600 bp in length, was retrieved from the
Manihot esculenta v6.1 genome in the phytozome v13 (
https://phytozome-next.jgi.doe.gov/ (accessed on 24 July 2023)) database. Specific primers were designed using the coding region sequence, and the primers are shown in
Table S2. Meanwhile, the reverse transcription product cDNA of the SC9 cassava leaves was used as the amplification template for PCR amplification. The reaction conditions of PCR amplification are shown in
Figure S4.
4.5. Transient Gene Expression in N. benthamiana Leaves
The vector utilized for subcellular localization of MebHLH149 protein was pNC-Green-SubN (GFP). The Nimble Cloning method was employed to amplify the target gene fragment, with 20 bp of universal adapter sequences appended to both termini of the gene-specific primers. The upstream primer sequence was 5′-agtggtctctgtccagtcct-3′, and the downstream primer sequence was 5′-ggtctcagcagaccacaagt-3′. Plasmids harboring the pCE2 TA/Blunt-Zero-
MebHLH149 recombinant vector were used as templates for amplification. Gel extraction was carried out for positive bands obtained from amplification. The primers used for amplification are presented in
Table S2. The recovered products were combined with Nimble Mix and an appropriate quantity of pNC-Green-SubN (GFP) plasmid, and the mixture was incubated in a PCR machine at 50 °C for 50 min before being placed on ice for transformation into
Escherichia coli.Ultimately, plasmids were extracted from the bacterial liquid with correct sequencing and then transformed into Agrobacterium GV3101 Psoup-p19. Suspensions of A. tumefaciens harboring the recombinant plasmid pNC-Green-SubN-MebHLH149 (GFP) and the empty vector pNC-Green-SubN (GFP) were independently injected into the abaxial side of N. benthamiana leaves. After one day of dark incubation followed by two days of weak light incubation, fluorescence signals were observed under a confocal laser scanning microscope (LEICA, Wetzlar, Germany).
4.6. Virus-Induced Gene Silencing in Cassava
The cassava common mosaic virus (CsCMV) VIGS vector was used to construct the
MebHLH149 silencing vector pCsCMV-
MebHLH149. The pCsCMV-NC vector utilized in this experiment was developed by Dr. Yan Pu and colleagues at the Chinese Academy of Tropical Agricultural Sciences [
33]. First, specific 300 bp fragments of
bHLH149 were selected using the online SGN VIGS Tool (
https://vigs.solgenomics.net/ (accessed on 31 July 2023)). Primers were designed based on this sequence for amplification and connection to the target vector (pCsCMV-NC). Nimble Cloning was employed to ligate the target fragment and the pCsCMV-NC vector, ultimately obtaining the pCsCMV-
MebHLH149 recombinant vector required for VIGS. Firstly, the plasmid was transformed into
Agrobacterium tumefaciens, and the bacterial suspension was subsequently inoculated into the leaves of the cassava variety SC9 that had grown for approximately 40 days. The cassava leaves of the negative control group was inoculated with pCsCMV-NC, and those of the positive control group were inoculated with pCsCMV-
ChlI345, and the experimental group was inoculated with
Agrobacterium tumefaciens of pCsCMV-
MebHLH149.
Subsequently, on the 26th day post-infiltration, the silencing efficiency in test plants was detected by real-time quantitative PCR. MebHLH149-silenced plants were inoculated with Xpm CHN01 on their newly emerged leaves, and their phenotypic alterations were observed at 0d, 1d, and 2d to analyze gene expression and disease resistance in cassava leaves.
4.7. Genetic Transformation of the MebHLH149 Gene
MebHLH149 gene overexpression was accomplished using the pNC-Cam1304-MCS35S vector. In this experiment, the
MebHLH149 gene was cloned into the pNC-Cam1304-MCS35S vector using the same method as described in
Section 4.5. Once the pNC-Cam1304-MCS35S-
MebHLH149 recombinant vector was constructed, it was transformed into
Agrobacterium strain LBA4404. The
Agrobacterium containing pNC-Cam1304-MCS35S-
MebHLH149 and the empty vector pNC-CamM1304-MCS35S was cultivated at 28 °C until the OD
600 value ranged from 0.75 to 1.0. Then, the
MebHLH149 gene was transformed into the cassava friable embryogenic callus (FEC). To view the workflow of the
MebHLH149 gene infecting cassava FEC, please refer to
Figure S2. The composition of the medium used for tissue culture and transformation experiments referred to the configuration method of Nyaboga et al. [
30].
4.8. Screening of MebHLH149 Interacting Protein
To explore the interaction of proteins with MebHLH149, we employed the online String database (
https://cn.string-db.org/cgi/input?sessionId=bYYPxcIVb7Mw/ (accessed on 7 December 2023)) to predict a protein–protein interaction network involving the MebHLH149 protein (see
Figure S3). We selected high-scoring proteins containing the bHLH domain, MePRE5 and MePRE6, for subsequent validation.
4.9. Y2H Validation of Protein–Protein Interactions
The vectors employed in this experiment were pNC-GADT7 (AD) and pNC-GBKT7 (BD). The AD domain is derived from the transcription factor CAL4 and is located in the CAL4 activation domain at the C-terminal. Similarly, the BD domain is also from the transcription factor CAL4 and is located in the CAL4 DNA binding domain at the N-terminal. Firstly, the nucleotide sequence coding for MebHLH149 was cloned into the pNC-GADT7 vector, while the nucleotide sequences coding for MePRE5 and MePRE6 were cloned into the pNC-GBKT7 vector. The amplification and ligation of the gene fragments and subcellular vector construction were performed according to the methods described in
Section 4.5.
Prior to performing co-transformation, self-activation assays of the MebHLH149, MePRE5, and MePRE6 proteins were primarily conducted to observe whether there was any self-activation phenomenon. Subsequently, six groups of recombinant plasmids, namely AD + BD, AD-MebHLH149 + BD, BD-PRE5 + AD, BD-PRE5 + AD-MebHLH149, BD-PRE6 + AD, and BD-PRE6 + AD-MebHLH149, were co-transformed into the AH109 yeast competent cells. After the transformation, 20–50 μL were taken, respectively, and spread onto the SD/-Trp-Leu solid medium. When single colonies grew, the single colonies were picked and transferred to the SD/-Trp-Leu liquid medium for activation. The cells were centrifuged and the precipitate was re-suspended and diluted in four gradients according to the gradient dilution method and dropped onto the SD/-Trp-Leu and SD/-Trp-Leu-Ade-His media. The cultures were inverted and incubated at 29 °C.
4.10. BiFC Validation of Protein–Protein Interactions
The pNC-BiFC-Enn and pNC-BiFC-Ecc vectors were used in these experiments. To verify whether the MebHLH149 protein interacts with the PRE5 and PRE6 proteins, the MebHLH149 coding sequence was cloned into the pNC-BiFC-Enn vector, while the MePRE5 and MePRE6 coding sequences were cloned into the pNC-BiFC-Ecc vector. The constructed vectors were transformed separately into A. tumefaciens GV3101 Psoup-p19. A bacterial suspension was prepared using infiltration suspension (10 mM MES, 10 mM MgCl2, 100 µM acetosyringone) and adjusted to OD600 = 0.5. Equal volumes of the mixed bacterial suspension were prepared and left to stand in the dark for approximately 2 h. Using sterile disposable syringes, the mixed bacterial suspensions were injected separately onto the underside of N. benthamiana leaves. At 3 dpi, after staining with DAPI, the YFP signal was detected using confocal laser scanning microscopy (TCS SP8, Leica), and the in vivo interactions of MebHLH149, MePRE5, and MePRE6 were analyzed.
4.11. Statistical Analysis
All bar charts in this study were created based on data from three biological replicates, and normality tests were performed using Prism 8.0.2 software (
https://www.graphpad.com/ (accessed on 29 April 2023)). Significant differences were assessed using ANOVA and Dunnett’s test.
4.12. Accession Numbers
The sequence data used in this study can be found in the Phytozome v13 and NCBI (
https://www.ncbi.nlm.nih.gov/ (accessed on 25 July 2023)) online databases. The accession numbers for the
MebHLH149 gene and related research genes are presented in
Table S4.