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Article

Functional Analysis of MaWRKY24 in Transcriptional Activation of Autophagy-Related Gene 8f/g and Plant Disease Susceptibility to Soil-Borne Fusarium oxysporum f. sp. cubense

by
Guoyin Liu
,
Hongqiu Zeng
,
Xiang Li
,
Yunxie Wei
and
Haitao Shi
*
Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, College of Tropical Crops, College of Forestry, Hainan University, Haikou 570228, China
*
Author to whom correspondence should be addressed.
Pathogens 2019, 8(4), 264; https://doi.org/10.3390/pathogens8040264
Submission received: 30 October 2019 / Revised: 16 November 2019 / Accepted: 20 November 2019 / Published: 25 November 2019
(This article belongs to the Special Issue Plant Resistance Induced by Microorganisms and Pathogens)
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Abstract

:
WRKYs play important roles in plant development and stress responses. Although MaWRKYs have been comprehensively identified in the banana (Musa acuminata), their in vivo roles and direct targets remain elusive. In this study, a transcript profile analysis indicated the common regulation of MaWRKYs transcripts in response to fungal pathogen Fusarium oxysporum f. sp. cubense (Foc). Among these MaWRKYs, MaWRKY24 was chosen for further analysis due to its higher expression in response to Foc. The specific nucleus subcellular location and transcription activated activity on W-box indicated that MaWRKY24 was a transcription factor. The correlation analysis of gene expression indicated that MaWRKYs were closely related to autophagy-associated genes (MaATG8s). Further analysis showed that MaWRKY24 directly regulated the transcriptional level of MaATG8f/g through binding to W-box in their promoters, as evidenced by quantitative real-time Polymerase Chain Reaction (PCR), dual luciferase assay, and electrophoretic mobility shift assay. In addition, overexpression of MaWRKY24 and MaATG8f/g resulted in disease susceptibility to Foc, which might be related to the activation of autophagic activity. This study highlights the positive regulation of MaWRKY24 in transcriptional activation of autophagy-related gene 8f/g in the banana and their common roles in disease susceptibility to soil-borne Foc, indicating the effects of MaWRKY24 on autophagy and disease susceptibility.

1. Introduction

In recent years, the widespread soil-borne fungus Fusarium oxysporum (Fo) has caused vascular wilt disease and serious yield loss in crops [1,2]. Fusarium wilt of the banana (Panama disease) caused by soil-borne Fusarium oxysporum f. sp. cubense (Foc) is one of the most destructive [3,4,5,6]. What is worse, the soil-borne disease spreads rapidly, and no sustainable control method has been developed so far, thus posing a great threat to banana production all over the world [3,6,7,8]. Based on different banana hosts, at least three strains of Foc have been identified: Foc strain 1 (Foc 1), Foc strain 2 (Foc 2), and Foc strain 4 (Foc 4) [4,6]. The initial symptom of banana wilt is blister damage on the leaves, followed by progressive leaf yellowing from the lower leaves to the upper leaves in response to Foc infection [8]. Unfortunately, although some differently expressed genes have been identified in response to Foc infection [2], their roles remain unclear in vivo. Therefore, functional analysis of Foc-regulated genes might be helpful to better understand the molecular processes related to disease resistance and develop a disease-resistant variety through genetic breeding.
WRKY transcription factors are defined based on their DNA binding domain with WRKY amino acid sequences of 60 residues in the N-terminus. Moreover, they have an atypical zinc finger structure of Cx4-5Cx22-23HxH or Cx7Cx23HxC in the C-terminus [9,10,11]. WRKY transcription factors are widely involved in plant development and stress responses. On the one hand, WRKYs can be served as activators or repressors to modulate various plant processes [12,13]. On the other hand, WRKYs can interact with other proteins, such as mitogen-activated protein (MAP) kinase (MAPK) [10,14,15,16], MAP kinase kinase kinase (MEKK) [10,16,17], calmodulin [10], and histone deacetylases (HDAs) [10]. For example, the phosphorylation of WRKY7/8/9/11 by MAPK accelerates WRKY-dependent respiratory burst oxidase homolog (RBOHB) expression via binding to the cognate W-box in the promoter, resulting in ROS burst in Nicotiana benthamiana [18]. Interestingly, AtWRKY53 is phosphorylated by MEKK1; this may increase the binding of AtWRKY53 to its own promoter and regulate senescence [10].
WRKYs have been widely investigated in plants. For example, CsWRKY31 (Camellia Sinensis, Cs) and CsWRKY48 can inhibit the transcriptions of biosynthesis-related genes leucocyanidin reductase (LAR), dihydroflavonol-4-reductase (DFR), and caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) through binding to the W-box elements, served as negative regulators of O-methylated catechin biosynthesis [12]. WRKYs can also regulate leaf senescence by interacting with DELLA protein repressor of ga1-3-LIKE1 (RGL1) in the gibberellin signaling pathway [19], the epithio specifying senescence regulator (ESR/ESP) in salicylic acid (SA), and the jasmonic signaling pathway [20], respectively. Furthermore, the ectopic expression of VqWRKY52 (Vitis quinquangularis, Vq) in Arabidopsis thaliana enhances the resistance to powdery mildew and Pseudomonas syringae pv. tomato (Pst) DC3000 [21]. Similarly, SlWRKY8 (Solanum lycopersicum, Sl) is positively involved in disease resistance against Pst DC3000 via activating the transcription levels of the SlPR1a1 and SlPR7 [22]. Interesting, PtrWRKY18 (Populus trichocarpa, Ptr) and PtrWRKY35 activate pathogenesis-related (PR) genes, and increase the resistance to the biotrophic pathogen Melampsora [23]. In the banana, MaWRKY69 and MaWRKY92 are highly upregulated in the susceptible cultivar, but downregulated in the resistant cultivar after infection by the root lesion nematode Pratylenchus coffeae [24]. MaWRKY71-overexpressing transgenic bananas have increased salt and oxidative stress resistance compared to wild-type bananas, with no effect on the disease resistance against Foc [25]. Additionally, MaWRKY1/2 can directly bind to the promoters of MaPR1-1, MaPR2, and MaPR10c to regulate SA- and methyl jasmonate-induced pathogen resistance [10,26]. A recent study showed that MaWRKY31, MaWRKY33, MaWRKY60, and MaWRKY71 directly bind to the W-box elements in the promoters of 9-cis-epoxycarotenoid dioxygenase 1 (MaNCED1) and MaNCED2 and activate their expression, thereby regulating abscisic acid-induced cold tolerance in banana fruit [27]. Although MaWRKYs are comprehensively identified in the banana, the in vivo roles remain elusive [9,24], and need to be investigated further.
In this study, gene expression analysis revealed that some MaWRKYs might be involved in the defense response to fungal pathogen Foc. Further analysis revealed the subcellular location and transcription-activated activity on the W-box (TTGACC/T) of MaWRKY24. Notably, the transcriptional activation of MaWRKY24 on several autophagy-associated genes (ATGs, MaATG8f/g) and their roles in plant disease susceptibility to soil-borne Foc were also highlighted. This study provided novel insight into MaWRKY24i mediated autophagy in soil-borne disease susceptibility.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

The banana variety BaXi jiao (BX) was used in this study. Tissue culture seedlings of bananas from the Tropical Seedling Tissue Culture Center (Danzhou, Hainan, China) were cultivated in Murashige and Skoog (MS) in the greenhouse, with 12 h light/ dark cycles and 120–150 µmol quanta m-2 s-1 irradiance at 26 °C.
The Arabidopsis seeds were sterilized by 70% ethanol for 1 min, followed by 4% NaClO for 5 min, and then washed at least five times with sterile, distilled water. After being kept at 4 °C in the dark for 24 h, they were placed on plates containing MS medium with 2% sucrose (pH 5.8). Arabidopsis seedlings were cultivated in a greenhouse (12 h light/ dark cycles and 120–150 µmol quanta m-2 s-1 irradiance at 22 °C).
Foc 4 was cultured on potato dextrose agar (PDA) medium for 7 d at 28 °C in the dark, and then fresh Foc 4 was washed out using sterilized water. The washed Foc 4 solution was filtered by sterilized six-layer gauze to remove the mycelium, and the spore solution concentration was adjusted to 5 × 106 spores/mL for pathogen inoculation [6].

2.2. Phylogenetic Analysis of MaWRKYs

Obtaining sequences of 147 MaWRKYs and 82 AtWRKYs from Phytozome v12.1 (https://phytozome.jgi.doe.gov/pz/portal.html) and the Arabidopsis Information Resource (TAIR) version 10 (https://www.arabidopsis.org/), respectively, then the corresponding phylogenetic tree was structured by using Clustalx 1.83 and MEGA5.05 [28].

2.3. RNA Isolation and Quantitative Real-Time PCR (qRT-PCR)

RNA isolation was performed using a kit (DP441, TIANGEN, Beijing, China), and qRT-PCR was performed using the LightCycler® 96 Real-Time PCR System (Roche, Basel, Switzerland), as Wei et al. [29] previously described. qRT-PCR profiles were determined following the protocol: 95 °C for 10 min, 45 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 20 s. Based on the Ct values, the corresponding gene expression levels were analyzed with the comparative 2(-ΔΔCt) method [23]. The primers are listed in Table S1.

2.4. Subcellular Localization Analysis

The coding sequences of MaWRKY24 were cloned into pEGAD vector [30] to form the constructions of 35S::GFP-MaWRKY24. The primers are listed in Table S2. Subsequently, the GV3101 strains harboring an empty vector or the recombinant plasmids were syringe-infiltrated into Nicotiana benthamiana leaves, as previously described by Sparkes et al. [31]. Two days post-infiltration, the GFP signals of 35S::GFP and 35S::GFP-MaWRKY24 and 4′, 6-diamidino-2-phenylindole (DAPI)-stained cell nuclei were detected via a confocal laser-scanning microscope (TCS SP8, Leica, Heidelberg, Germany).

2.5. Dural Luciferase (LUC) Assay through Transient Expression

5×TTGACC/T (W-box) and the promoter sequences of MaATG8s were cloned into the pGreenII 0800-LUC vector to form the constructions of 35S::REN-5×W-box/proMaATG8s-LUC. The primers are listed in Table S2. After 12 h of transformation in leaf protoplasts, as Yoo et al. [32] previously described, the LUC and REN were quantified in the transformed protoplasts using a dual luciferase reporter gene assay kit (RG027, Beyotime, Haimen, Jiangsu, China).

2.6. Chromatin Immunoprecipitation Quantitative Real-Time PCR (ChIP-qPCR)

Banana protoplast isolation was performed according to Sagi et al. [33]. The banana nucleus was extracted using Plant Nuclei Isolation/Extraction Kit (CELLYTPN1, Sigma, Missouri, USA) according to the instruction. Then each sample was divided into two parts, protein A/G (C40091707, GenScript) and GFP antibody (G1546, Sigma) or IgG (A4416, Sigma) was added, and the solution was mixed and incubated for 6 h at 4 °C. DNA isolation was performed using a kit (32817KC1, AXYGEN), and qRT-PCR was performed. The primers are listed in Table S1.

2.7. Electrophoretic Mobility Shift Assay (EMSA)

The coding sequences of MaWRKY24 was cloned into the pET28a vector to form the constructions of pET28a-MaWRKY24. The primers are listed in Table S1. The BL21 strain harboring pET28a-MaWRKY24 was induced by 1 mM IPTG for protein expression and purification (His-tag Protein Purification Kit, P2226, Beyotime, Haimen, Jiangsu, China), and the synthesized double-stranded probes were used for EMSA, as Ream et al. [34] previously described.

2.8. Generation of Transgenic Plants and Observation of the Autophagosome

The coding sequences of MaATG8f/g have been cloned into the pEGAD vector [30] as per a previous work [29]. Then the recombinant plasmids (35S::GFP-MaWRKY24, 35S::GFP-MaATG8f and 35S::GFP-MaATG8g) were transformed into Agrobacterium tumefaciens GV3101, which was further used to obtain transgenic Arabidopsis plants via the floral dip method [35,36]. The positive transgenic plants were chosen by Basta resistance and confirmed by PCR. For the observation of autophagosomes, 14-day-old seedlings of wild-type (WT) and MaATG8f/g-overexpressing lines were transferred into a MS medium with or without Foc 4 spore solution for 8 h. Thereafter, the GFP fluorescence in Arabidopsis roots was detected using biotechnical microscopy (DM5000, Leica, Wetzlar, Germany).

2.9. Statistical Analysis

In this study, at least three biological replicates were carried out for all experiments, and the average means ± SD are shown. After comprehensive analysis by Duncan’s range test using Statistical Analysis System (SAS) v9.4 software (SAS Instituteinc, North Carolina, USA), asterisks (*) show significant difference at p < 0.05.

3. Results

3.1. Evolutionary Analysis and Expression Profiles of MaWRKYs

To analyze the evolutionary relationships between MaWRKYs and AtWKRYs, an unrooted neighbor-joining phylogenetic tree was constructed based on the predicted amino acid sequences. As shown in Figure 1, a neighbor-joining phylogenetic tree, with five groups, was constructed to investigate the evolution between 147 MaWRKYs and 82 AtWRKYs. The groups contained 39, 14, 42, 27, and 25 MaWRKYs. Among them, MaWRKY24 belonged to the second group, and showed high homology with AtWRKY48, AtWRKY49, and AtWRKY54. Moreover, both AtWRKY48 and AtWRKY54 are negative regulators in plant basal defense response [37,38]. Based on previously published data [2], we analyzed the transcript profiles of MaWRKYs in banana roots’ response to Foc 1/4 infection. In brief, 60 of 147 MaWRKYs were significantly induced in response to Foc infection, with at least 2-fold changes (Figure 2). Some MaWRKYs, such as MaWRKY28/73/145, were generally upregulated by Foc 1/4 infection, while some MaWRKYs (MaWRKY76, 82, 84, 103, and so on) were commonly downregulated by Foc 1/4 infection (Figure 2). Interestingly, MaWRKY24 was significantly upregulated at 3 and 51 h, but downregulated at 27 h by Foc 1/4. According to Li et al. [2], 27 h post-inoculation, spores and hyphae are attached to the banana roots inoculated with Foc ¼; 51 h post-inoculation, hyphae spread into the vascular tissues of the roots infected with Foc 1/4. Therefore, the induction of MaWRKY24 might be involved in early and later stages of banana‒Foc 1/4 interaction, and the dual transcriptional changes of MaWRKY24 also indicate its precise modulation in the banana‒Foc 1/4 interaction. In addition, MaWRKY24 showed high homology with AtWRKY48 and AtWRKY54, which are negative regulators of plant basal defense response [37,38]. Therefore, MaWRKY24 was selected for further functional analysis.

3.2. Subcellular Localization and Transcriptional Activated Activity of MaWRKY24

In the transient N. benthamiana leaves, the fluorescence of 35S::GFP was located in both the cytoplasm and nucleus, while that of 35S::GFP-MaWRKY24 was specifically colocalized with DAPI-stained nucleus (Figure 3). As transcription factors, plant WRKYs are widely recognized as binding to the W-box (5’-TTGACC/T-3’). Using dual LUC assay in plant leaf protoplasts, the overexpression of MaWRKY24 significantly activated the LUC of 5×W-box-pGreenII 0800-LUC (Figure 4). Therefore, the specific nucleus subcellular location and transcription-activated activity on the W-box indicated that MaWRKY24 was a transcription factor.

3.3. Overexpression of MaWRKY24 Regulated Expression Level of MaATG8s

In previous studies, we found that MaATG8s were also commonly regulated by Foc 1 and Foc 4 [29]. Interestingly, the promoters of some MaATG8s are widely distributed with the W-box. To investigate the relationship between these MaWRKYs and MaATG8s, we performed a cluster analysis of the correlation between MaWRKYs and MaATG8s expression. The result showed that the expression of MaWRKY24 was most closely related to that of MaATG8f/g (Figure S1). Consistently, we found that the relative transcriptional levels of MaATG8f/g were significantly upregulated in MaWRKY24-overexpressing protoplasts (Figure 5), indicating that MaWRKY24 could activate the expression of MaATG8f/g.

3.4. MaWRKY24 were Transcriptional Activators of MaATG8f/g

In addition, we investigated the direct effect of MaWRKY24 overexpression on the promoter activities of MaATG8f/g. Dual LUC assay in leaf protoplasts showed that overexpression of MaWRKY24 significantly activated the LUC of MaATG8f/g promoters (Figure 6A). To determine whether MaWRKY24 combined with the W-box element, ChIP-qPCR analysis was performed. The result showed that the relative enrichment of promoter regions of MaATG8f/g with W-box was higher than that of control (Figure 6B).
To confirm the direct binding of MaWRKY24 to the promoter region of MaATG8f/g with the W-box, the protein of MaWRKY24 was induced and purified for EMSA analysis. The result confirmed that MaWRKY24 direct binding to the corresponding probes containing W-box in MaATG8f/g promoters in vitro (Figure 7). In summary, these results suggested that MaWRKY24 bound to the W-box regions in the promoter of MaATG8f/g and served as a transcriptional activator of MaATG8f/g.

3.5. Overexpression of MaWRKY24 and MaATG8f/g Negatively Regulate Plant Disease Susceptibility to Foc 4

To further study the roles of MaWRKY24 and MaATG8f/g in response to Foc 4 infection, 14-day-old MaWRKY24 and MaATG8f/g overexpressing Arabidopsis seedlings were grown under control and Foc 4 infection conditions. Under control conditions, there were no significant differences in phenotype between the WT and transgenic lines (Figure 8A–B). Under Foc 4 infection, both MaWRKY24- and MaATG8f/g-overexpressing Arabidopsis seedlings showed worse growth and lower chlorophyll (a+b) content than WT (Figure 8A–B). Therefore, overexpression of MaWRKY24 and MaATG8f/g might negatively regulate plant disease susceptibility to Foc 4 infection in Arabidopsis.
To investigate whether Foc 4 affected autophagy in Arabidopsis roots, the roots of WT, 35S::GFP and 35S:GFP-MaATG8f/g seedlings were infected with a Foc 4 spore solution for 8 h. The green fluorescent spots indicated that autophagosomes were stronger after Foc 4 infection in MaATG8f/g transgenic Arabidopsis, but not in the WT and the 35S::GFP transgenic line (Figure 9). The results indicated that Foc 4 might induce the formation of autophagosomes.

4. Discussion and Conclusions

Although the banana is one of the most popular fresh fruits in the world, it is seriously affected by Foc in subtropical and tropical areas [2]. Furthermore, no strong and continuous disease-resistant banana variety is effective in the banana cultivation [39]. Therefore, it is essential to construct strong disease-resistant banana varieties through molecular and genetic breeding.
WRKY transcription factors are widely involved in plant stress responses [18,40,41,42,43,44]. In plants, most WRKY transcription factors are activated by plant‒pathogen interaction, and regulate multiple downstream genes via binding to W-box elements in the promoter [45,46,47]. Herein, we found that 60 of 147 MaWRKYs were significantly affected by Foc infection. Additionally, the overexpression of MaWRKY24 negatively regulates plant disease susceptibility to Foc 4. In Arabidopsis, flg22 induces the transcripts of AtWRKY18, AtWRKY33, and AtWRKY40, resulting in the activation of hundreds of genes with W-box elements [45]. AtWRKY8 has a positive effect on the resistance to Pseudomonas syringae, B. cinerea, and salinity stress [48,49]. Consistently, SlWRKY8 functions as a positive regulator in resisting Pst DC3000 [22]. However, AtWRKY18 and AtWRKY40 act as negative resistance against the obligate hemibiotrophic fungus Golovinomyces orontii [50], and PtrWRKY40 also negatively regulates disease resistance against Dothiorella gregaria in the poplar [51]. Similarly, overexpression of GhWRKY25 enhances disease susceptibility to Botrytis cinerea by inhibiting the expression of SA or ethylene signaling-related genes [52]. It is widely known that the conservation of the WRKY domain can recognize and bind with the W-box [10,53]. We also found that the overexpression of MaWRKY24 could significantly recognize and bind with the W-box. For example, PcWRKY1, a transcriptional activator, mediates fungal elicitor-induced gene expression by binding to W-box elements [54]. HvWRKY38 binding to W-box elements are involved in cold and drought response [55]. In this study, MaWRKY24 had significant effects on the W-box, together with the specific nucleus subcellular location, indicating that it is a real WRKY transcript factor.
As the key regulator of cellular homeostasis, autophagy is a transport pathway that mediates the transfer and degradation of cytoplasmic materials [56]. Briefly, autophagy breaks down the damaged cytoplasmic constituents in a cell and recycles the cellular cytoplasmic components [57]. Autophagy is coordinated by evolutionarily conserved ATGs that are essential for biotic and abiotic stress responses [58,59,60,61,62]. For example, autophagic activity is induced by necrotrophic fungal pathogens [63,64]. Herein, the green fluorescence spots in MaATG8f/g-overexpressing lines were stronger after Foc 4 spore solution treatment compared to the control, indicating the activation of autophagosomes and autophagic activity by Foc 4. Meanwhile, MaATG8f/g played a negative role in disease susceptibility to Foc 4. Recently, we have highlighted the effects of MaATG8s on hypersensitive-like cell death and immune responses, which are directly related to autophagy [29]. Moreover, ATG8s are central parts of the latter process among autophagy-related proteins [58]. The results showed that the expression of MaWRKY24 was closely related to that of MaATG8f/g. MaWRKY24 was the transcriptional activator of MaATG8f/g, due to the direct binding of MaWRKY24 to the W-box in the promoter of MaATG8f/g. AtWRKY33 interacts with ATG18a and both are involved in plant responses to necrotizing trophic pathogens [65]. Previous studies have shown that the crosstalk of WRKYs and ATGs as well as autophagy may be involved in plant resistance to necrotrophic pathogens and bacterial pathogens [65,66,67].
This study highlights the positive regulation of MaWRKY24 in the transcriptional activation of MaATG8f/g and their common roles in plant disease susceptibility to Foc 4, indicating the correlation between MaWRKY24, autophagy, and disease susceptibility (Figure 10).

Supplementary Materials

The following are available online https://www.mdpi.com/2076-0817/8/4/264/s1. Table S1: The primers were used for the quantitative real time PCR. Table S2: The primers were used for the vector construction. Figure S1: Cluster analysis of correlation between MaWRKYs and MaATG8s expression.

Author Contributions

Formal analysis, H.Z.; Investigation, G.L. and X.L.; Methodology, H.Z., Y.W. and H.S.; Project administration, H.S.; Writing—original draft, G.L.; Writing—review & editing, H.S.; Funding acquisition, H.S.

Acknowledgments

This research was supported by startup funding and the Scientific Research Foundation of Hainan University (No. kyqd1531).

Conflicts of Interest

All authors declare no conflict of interest.

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Figure 1. A neighbor-joining phylogenetic tree was constructed to investigate the evolution of MaWRKYs and AtWRKYs. The green and red shapes show AtWRKYs and MaWRKYs, respectively. The phylogenetic tree was established on the basis of the coding sequence using Clustalx 1.83 and MEGA5.05. The red asterisk indicates the location of MaWRKY24.
Figure 1. A neighbor-joining phylogenetic tree was constructed to investigate the evolution of MaWRKYs and AtWRKYs. The green and red shapes show AtWRKYs and MaWRKYs, respectively. The phylogenetic tree was established on the basis of the coding sequence using Clustalx 1.83 and MEGA5.05. The red asterisk indicates the location of MaWRKY24.
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Figure 2. The significant relative transcription levels of MaWRKYs in response to Foc 1 and Foc 4. The values of transcriptomic data of corresponding genes were downloaded from the Supplementary Table S4 in Li et al. [2], and have also described in the Supplementary Table 5 in Goel et al. [9]. In the assay, banana roots were inoculated by the control or Foc 1 or Foc 4 for 3 h, 27 h, or 51 h. A heatmap of gene expression profile was constructed using CLUSTER and Java Treeview. The red asterisk indicates the location of MaWRKY24.
Figure 2. The significant relative transcription levels of MaWRKYs in response to Foc 1 and Foc 4. The values of transcriptomic data of corresponding genes were downloaded from the Supplementary Table S4 in Li et al. [2], and have also described in the Supplementary Table 5 in Goel et al. [9]. In the assay, banana roots were inoculated by the control or Foc 1 or Foc 4 for 3 h, 27 h, or 51 h. A heatmap of gene expression profile was constructed using CLUSTER and Java Treeview. The red asterisk indicates the location of MaWRKY24.
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Figure 3. Subcellular localization of MaWRKY24. The GFP signals of 35S::GFP and 35S::GFP-MaWRKY24 and DAPI-stained cell nuclei are shown. Bar = 25 μm.
Figure 3. Subcellular localization of MaWRKY24. The GFP signals of 35S::GFP and 35S::GFP-MaWRKY24 and DAPI-stained cell nuclei are shown. Bar = 25 μm.
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Figure 4. The transcriptional activated activity of MaWRKY24. In the dual LUC assay in plant leaf protoplasts, 35S::GFP and 35S::GFP-MaWRKY24 were used as the effectors, and 35S::REN-5×W-box-LUC was used as the reporter. Asterisks (*) show significant difference at p < 0.05.
Figure 4. The transcriptional activated activity of MaWRKY24. In the dual LUC assay in plant leaf protoplasts, 35S::GFP and 35S::GFP-MaWRKY24 were used as the effectors, and 35S::REN-5×W-box-LUC was used as the reporter. Asterisks (*) show significant difference at p < 0.05.
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Figure 5. The effect of MaWRKY24 overexpression on the relative transcription levels of MaATG8f/g in leaf protoplasts. Asterisks (*) show significant difference at p < 0.05.
Figure 5. The effect of MaWRKY24 overexpression on the relative transcription levels of MaATG8f/g in leaf protoplasts. Asterisks (*) show significant difference at p < 0.05.
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Figure 6. MaWRKY24 as a transcriptional activator of MaATG8f/g. (A) In the dual LUC assay, 35S::GFP and 35S::GFP-MaWRKY24 were used as the effectors, and 35S::REN-pMaATG8s-LUC was used as the reporter in plant leaf protoplasts. (B) Analysis of the relative enrichment of the MaATG8s promoter by ChIP-qPCR in banana leaf protoplasts. Asterisks (*) show significant difference at p < 0.05.
Figure 6. MaWRKY24 as a transcriptional activator of MaATG8f/g. (A) In the dual LUC assay, 35S::GFP and 35S::GFP-MaWRKY24 were used as the effectors, and 35S::REN-pMaATG8s-LUC was used as the reporter in plant leaf protoplasts. (B) Analysis of the relative enrichment of the MaATG8s promoter by ChIP-qPCR in banana leaf protoplasts. Asterisks (*) show significant difference at p < 0.05.
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Figure 7. Assay of the interaction between MaWRKY24 and MaATG8f/g promoters. (A) MaWRKY24-pET28a induced expression by SDS-PAGE. The red line indicates the induced recombinant protein. (B) MaWRKY24-pET28a was purified. 1‒6 indicate the first to sixth tubes of the collected purified protein by elution. The red line indicates the corresponding induced recombinant protein. (C) The probe sequences of the wild type and the mutation. (D) The binding of MaWRKY24 to the W-box of MaATG8f/g promoters through EMSA. The arrow and square frame indicate the probe‒protein complex.
Figure 7. Assay of the interaction between MaWRKY24 and MaATG8f/g promoters. (A) MaWRKY24-pET28a induced expression by SDS-PAGE. The red line indicates the induced recombinant protein. (B) MaWRKY24-pET28a was purified. 1‒6 indicate the first to sixth tubes of the collected purified protein by elution. The red line indicates the corresponding induced recombinant protein. (C) The probe sequences of the wild type and the mutation. (D) The binding of MaWRKY24 to the W-box of MaATG8f/g promoters through EMSA. The arrow and square frame indicate the probe‒protein complex.
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Figure 8. Phenotypes of WT and transgenic seedlings in response to Foc 4 infection. (A) The phenotypes of seedlings under control and Foc 4 infection conditions. Fourteen-day-old seedlings of WT, MaWRKY24- and MaATG8f/g-overexpressing lines were without or infected with Foc 4 spore solution for 7 d and then the phenotype was observed. Bar = 1 cm. (B) Relative chlorophyll (a + b) of WT and transgenic seedlings. Asterisks (*) show significant difference at p < 0.05.
Figure 8. Phenotypes of WT and transgenic seedlings in response to Foc 4 infection. (A) The phenotypes of seedlings under control and Foc 4 infection conditions. Fourteen-day-old seedlings of WT, MaWRKY24- and MaATG8f/g-overexpressing lines were without or infected with Foc 4 spore solution for 7 d and then the phenotype was observed. Bar = 1 cm. (B) Relative chlorophyll (a + b) of WT and transgenic seedlings. Asterisks (*) show significant difference at p < 0.05.
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Figure 9. The effect of Foc 4 on the formation of autophagosomes in Arabidopsis seedling roots. For the examination, 14-day-old seedlings of WT, 35S::GFP and MaATG8f/g overexpressing lines were transferred into MS medium with or without Foc 4 spores solution for 8 h and then the autophagosomes were examined. Autophagosomes were marked with circles. Bar = 100 μm.
Figure 9. The effect of Foc 4 on the formation of autophagosomes in Arabidopsis seedling roots. For the examination, 14-day-old seedlings of WT, 35S::GFP and MaATG8f/g overexpressing lines were transferred into MS medium with or without Foc 4 spores solution for 8 h and then the autophagosomes were examined. Autophagosomes were marked with circles. Bar = 100 μm.
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Figure 10. A model for the correlation between MaWRKY24 and autophagy in the banana.
Figure 10. A model for the correlation between MaWRKY24 and autophagy in the banana.
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Liu, G.; Zeng, H.; Li, X.; Wei, Y.; Shi, H. Functional Analysis of MaWRKY24 in Transcriptional Activation of Autophagy-Related Gene 8f/g and Plant Disease Susceptibility to Soil-Borne Fusarium oxysporum f. sp. cubense. Pathogens 2019, 8, 264. https://doi.org/10.3390/pathogens8040264

AMA Style

Liu G, Zeng H, Li X, Wei Y, Shi H. Functional Analysis of MaWRKY24 in Transcriptional Activation of Autophagy-Related Gene 8f/g and Plant Disease Susceptibility to Soil-Borne Fusarium oxysporum f. sp. cubense. Pathogens. 2019; 8(4):264. https://doi.org/10.3390/pathogens8040264

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Liu, Guoyin, Hongqiu Zeng, Xiang Li, Yunxie Wei, and Haitao Shi. 2019. "Functional Analysis of MaWRKY24 in Transcriptional Activation of Autophagy-Related Gene 8f/g and Plant Disease Susceptibility to Soil-Borne Fusarium oxysporum f. sp. cubense" Pathogens 8, no. 4: 264. https://doi.org/10.3390/pathogens8040264

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