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Article

Three Main Genes in the MAPK Cascade Involved in the Chinese Jujube-Phytoplasma Interaction

by
Zhiguo Liu
1,
Zhihui Zhao
1,
Chaoling Xue
2,
Lixin Wang
3,
Lili Wang
1,
Chunfang Feng
4,
Liman Zhang
2,
Zhe Yu
5,
Jin Zhao
2 and
Mengjun Liu
1,3,6,*
1
Research Center of Chinese Jujube, Hebei Agricultural University, Baoding 071001, China
2
College of Life Science, Hebei Agricultural University, Baoding 071001 China
3
College of Horticulture, Hebei Agricultural University, Baoding 071001, China
4
College of Forestry, Hebei Agricultural University, Baoding 071001, China
5
Forestry Bureau of Zhangbei County, Zhangjiakou 075000, China
6
Beijing Collaborative Innovation Center for Eco-enviromental Improvement with Forestry and Fruit Trees, Beijing 100000, China
*
Author to whom correspondence should be addressed.
Forests 2019, 10(5), 392; https://doi.org/10.3390/f10050392
Submission received: 7 March 2019 / Revised: 24 April 2019 / Accepted: 30 April 2019 / Published: 2 May 2019
(This article belongs to the Special Issue Roles and Interactions of Insects and Microbes in Forest Systems)
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Versions Notes

Abstract

:
Chinese jujube (Ziziphus jujuba Mill.) is an important economic forest species and multipurpose fruit tree in the family of Rhamnaceae. Phytoplasmas are significant prokaryotic pathogens, associated with more than 1000 plant diseases. Jujube witches’ broom disease (JWB) is a typical phytoplasma disease, caused by ‘Candidatus Phytoplasma ziziphi’. Mitogen-activated protein kinase (MAPK) cascades are highly universal signal transduction modules and play crucial roles in regulating innate immune responses in plants. Thus, in the current study, systematical expression profiles of 10 ZjMPK and 4 ZjMPKK genes were conducted in plantlets with JWB disease, plantlets recovered from JWB disease, the tissues showing different disease symptoms, and resistant/susceptible cultivars infected by JWB phytoplasma. We found that most ZjMPK and ZjMKK genes exhibited significant up- or down-regulation expression under phytoplasma infection, but the top three differentially expressed genes (DEGs) were ZjMPK2, ZjMKK2 and ZjMKK4, which showed the biggest times of gene’s significant difference expression in all materials. Based on STRING database analysis, ZjMKK2 and ZjMPK2 were involved in the same plant-pathogen interaction pathway, and Yeast two-hybrid screening showed that ZjMKK2 could interact with ZjMPK2. Finally, we deduced a pathway of jujube MAPK cascades which response to ‘Candidatus Phytoplasma ziziphi’ infection. Our study presents the first gene-family-wide investigation on the systematical expression analysis of MAPK and MAPKK genes in Chinese jujube under phytoplasma infection. These results provide valuable information for the further research on the signaling pathway of phytoplasma infection in Chinese jujube.

1. Introduction

Plant pathogenic phytoplasmas, members of the class Mollicutes, are non-helical, cell wall-less bacterial organisms that are confined in the phloem sieve elements of infected plants [1,2]. Phytoplasmas are important prokaryotic pathogens that are associated with more than 1000 plant species, including many important economically plants [3,4]. Recently, phytoplasma effectors (such as SAP54, SAP11 and TENGU) have been shown to modulate plant development, including indeterminate leaf-like flower development, morphology alternation and sterility in Arabidopsis plants [5,6,7]. These results indicate that phytoplasma effectors play an important role in phytoplasma pathogenesis.
Chinese jujube (Ziziphus jujuba Mill.), also known as Chinese date, is an important economic forest species and multipurpose fruit tree in the family of Rhamnaceae. It has been cultivated in China for up to 7000 years and has been introduced to 47 countries throughout Americas, southern and eastern Asia, Europe, and Australia [8]. Jujube witches’ broom (JWB) caused by ‘Candidatus Phytoplasma ziziphi’ [9], was a typical phytoplasma disease. Since 1980s, JWB has become one of the most serious and destructive disease in jujube cultivation and the initial mechanism of phytoplasma infection on Chinese jujube has been revealed [10].
Phytoplasmas duplicate exclusively inside phloem sieve elements of host plants and within insect vectors [11]. They are transmitted by phloem-sap-feeding insects in a circulative and persistent manner and by vegetative propagation of plants, in particular, by grafting [12]. Although axenic cultivation of grapevine yellows phytoplasma under microaerophilic growing conditions was reported [13], most phytoplasma strains are not successfully cultivated in vitro due to the complex media and strict anaerobic conditions [14]. So, interactions between phytoplasmas pathogen and host plants are hindered by the fact that phytoplasmas cultivation on artificial media is difficult, and they cannot be transmitted mechanically [15,16].
Mitogen-activated protein kinase (MAPK) cascades are highly universal signal transduction modules and play crucial roles in regulating innate immune responses in eukaryotes, including yeasts, animals and plants [17,18,19,20]. A typical MAPK cascade consists of three classes of hierarchically organized protein kinases, i.e., MAPK kinase kinase (MAPKKK/MEKK), MAPK kinase (MAPKK/MEK) and MAPK (MAPK/MPK) which are linked in various ways with upstream receptors and downstream targets [21]. Increasingly studies have clearly identified some complete MAPK cascades particularly in immune response [19,20]. For example, AtMEKK1-AtMKK4/5- AtMPK3/6-WRKY22/29 has been implicated in flagellin-mediated innate immune response [22]. Another MAPK cascade pathway AtMEKK1-AtMKK1/2-AtMPK4-MKS1/WRKY33 has been shown to negatively regulate plant immune signaling [23,24]. Orthologs of the MAPK cascades in Arabidopsis have the same function in other plant species, such as tobacco [25,26], rice [27], and cotton [28].
In a previous study, our group found that the expression level of most ZjMPK and ZjMPKK genes were down-regulated in jujube plantlets within phytoplasma infection [29]. Ye et al. [30] found that differentially expressed proteins of Chinese jujube under phytoplasma infection through iTRAQ proteomics method, which was related to MAPK signaling pathway. However, there is still uncertainty on the role of MAPK cascade pathways in the Chinese jujube-phytoplasma interaction.
In Chinese jujube, we have systematical and typical materials which could be used for investigating the molecular interaction mechanism between jujube and phytoplasma. The materials include plantlets with JWB disease, plantlets recovered from JWB disease, tissues showing different disease symptoms, and JWB-resistant/susceptible jujube cultivars [31]. Furthermore, our research group has identified the ZjMPK and ZjMPKK gene families at genome level [29]. Thus, in this study, expression analysis of ZjMPK and ZjMPKK genes were comprehensively investigated in diseased plantlets, recovered plantlets from JWB diseased and healthy plantlets. In the meanwhile, the temporal and spatial expression profiles of ZjMPK and ZjMPKK genes were carried out in tissues showing different disease symptoms and in JWB-resistant/susceptible cultivars [10] under phytoplasma infection. These results will provide valuable information for functional dissection of the important candidate genes and facilitate the further study on the mechanism of plant-phytoplasma interaction.

2. Results

2.1. Expression Patterns of ZjMPK and ZjMKK Genes in Healthy, JWB-diseased and Recovered Plantlets

To detect the JWB disease of the different jujube plantlets (Figure S1), the phytoplasma concentration was measured at the tissue levels in the healthy plantlets, diseased plantlets and recovered plantlets. DAPI staining showed no fluorescent spots in the sieve element (SE) of healthy plantlets and recovered plantlets, but the fluorescent spots formed a large bright circle in the SE of the diseased plantlets (Figure S2).
Furthermore, gene expression profiles under biotic stresses usually act as indicators of gene function. Therefore, according to above results, we investigated the expression patterns of 10 ZjMPK and 4 ZjMKK genes in healthy, JWB-diseased and recovered plantlets by qRT-PCR and try to find out the potential genes responding to phytoplasma infection. As shown in Figure 1, the expression level of 5 ZjMPKs (1, 5, 7, 8 and 9) and the ZjMKK2 genes in diseased plantlets were down regulated compared to healthy plantlets, especially ZjMPK1, ZjMPK5, ZjMPK7 and ZjMKK2 with moderate-high fold change values of 1.87, 1.62, 1.74 and 1.80, respectively. In the meanwhile, none of the 14 genes were up regulated in diseased plants when compared to healthy plants. In addition, the expression level of ZjMPK3, 4, 6, 10 and ZjMKK3 show none significant changes in all three materials which demonstrate these genes might not be involved in jujube-phytoplasma interaction. Moreover, when comparing the expression level of these 14 genes in recovered plantlets to the diseased plantlets, only ZjMPK5, ZjMPK8, ZjMPK9 and ZjMKK2 were up-regulated and these genes are down regulated in diseased plantlets (Figure 1) which could suggest that ZjMPK5, ZjMPK9 and ZjMKK2 were the potential genes involved in phytoplasma infection.

2.2. Expression Modules of ZjMPK and ZjMKK Genes in Tissues Showing Different JWB Disease Symptoms

Based on above results, most ZjMPK and ZjMKK genes have strong response in diseased and recovered plantlets, but whether these genes have the similar expression trend in different JWB disease symptoms tissues remains unclear. Therefore, the ZjMPK and ZjMKK genes expression modules were investigated in small leaves, phyllody and non-symptomatic leaves of diseased jujube at five stages. As shown in Table 1, the significant difference times of gene expression amount to 45 which include 21 times of up-regulation and 24 times of down-regulation, the differentially expressed genes (DEGs, the gene’s relative expression change more than two-fold) distribute to 9 ZjMPKs and 2 ZjMKKs. Among them, the top 2 DEGs of ZjMPK gene family were ZjMPK2 (10 times, up-regulated 9 times and down-regulated 1 time) and ZjMPK9 (7 times up-regulation). In ZjMKK gene family, ZjMKK2 was the most extinctive DEG with the 9 significant difference times include 6 times down-regulation and 3 times up-regulation.
Moreover, the differentially expressed genes (DEGs) had similar expression profiles in different JWB disease symptoms tissues of their own except ZjMPK2 (Figure 2 and Figure 3). Because diseased jujube sprout at the late April, witches’ broom disease could only be shown in young branches limited that in May we could not collect the samples of phyllody and small leaves, thus we did the expression level of corresponding gene analysis from June to October. In non-symptomatic leaves, ZjMPK2 showed a slight decrease in transcript abundance from June to August, but sharply increased at September, then strictly decreased. In phyllody, ZjMPK2 showed a significant down-regulation from June to July, and then markedly increased at August, but strictly decreased from September to October. In small leaves, the expression level of ZjMPK2 slightly increased from June to July, then sharply increased at August, but significantly decreased at following stages.
Base on the significant difference times of gene expression in three JWB diseased tissues at five stages, ZjMPK2 and ZjMKK2 were screened out as candidate genes in response to phytoplasma infection. As shown in Figure 3, it can be observed that ZjMKK2 expression was mainly down-regulated in diseased tissues from June to October, while ZjMPK2 expression was up-regulated.

2.3. Expression Profiles of ZjMPK and ZjMKK Genes in Resistant and Susceptible Cultivars

Furthermore, in order to understand the expression divergence of ZjMPK and ZjMKK genes in resistant and susceptible cultivars after infected by phytoplasma, the expression profiles of ZjMPK and ZjMKK genes were conducted. As shown in Figure 4 and Table S1, 8 of 10 ZjMPK genes and 3 of 4 ZiMKK genes shown significant difference expression in both resistant and susceptible cultivars. Among them, ZjMPK2 and ZjMKK4 have the biggest times of gene’s significant difference expression, such as ZjMPK2 (8 times, up-regulated 7 times and down-regulated 1 time) and ZjMKK4 (8 times, up-regulated 2 times and down-regulated 6 times). ZjMPK2 transcript abundance significantly increased (expression changes > 2-fold) in susceptible cultivar after phytoplasma infection. Meanwhile, ZjMKK4 showed a decreased transcript level of more than 2-fold in susceptible cultivar after phytoplasma infection. In contrast, ZjMKK4 was significantly up-regulated in resistant cultivar.

2.4. Candidate Genes Identification and Protein-protein Interaction Analysis

According to the significant difference times of gene expression in all materials, ZjMPK2, ZjMKK2 and ZjMKK4 were selected as candidate genes which might play crucial roles in response to phytoplasma infection.
Protein-protein interaction analysis showed that ZjMPK2 and ZjMKK2 were involved in the same plant-pathogen interaction pathway (Figure S3), and our present study showed that ZjMKK2 could interact with ZjMPK2 by yeast two hybrid analysis (Figure S4). The homologous proteins of ZjMPK2 and ZjMKK2 were AtMPK3 and AtMKK6, respectively. AtMPK3 have been demonstrated to be the significant regulators in response to pathogen infections in Arabidopsis. ZjMKK4 was homologous with AtMKK10 which involved in the regulation in biological process.

2.5. The Transcriptional Difference of Candidate Genes between Susceptible and Resistant Cultivars

In order to clarify the transcriptional difference of candidate genes between susceptible and resistant cultivars, the expression profiles of ZjMPK2, ZjMKK2 and ZjMKK4 were analyzed after phytoplasma infection, as it shown in Figure 5. We found that ZjMPK2 expression level in susceptible cultivar was generally higher than that in resistant cultivar after phytoplasma infection. However, ZjMKK2 and ZjMKK4 transcripts showed a reverse trend compared to ZjMPK2, in particular, regarding the expression level of ZjMKK4. The relative expression level of ZjMKK4 in susceptible cultivar was significantly lower than that in resistant cultivar.

3. Discussion

As the core pathway of signal transduction networks, MAPK cascades play vital roles in defense response in plants. Previously, we have identified the ZjMPK and ZjMKK genes family from jujube genomic database, a systematic bioinformatics analysis of all ZjMPK and ZjMKK genes has been studied, and the expression profiles of all ZjMPK and ZjMKK genes under biotic and abiotic stresses were also conducted [29]. All of these results demonstrate all ZjMPK and ZjMKK genes function importantly in the signaling transduction under biotic stress. In this study, the transcription level of ZjMPK and ZjMKK genes was systematically analyzed under phytoplasma stress. Finally, ZjMPK2, ZjMKK2 and ZjMKK4 were identified as candidate genes which response to JWB phytoplasma infection.
MAPK regulatory pathway genes play crucial roles in plant developmental processes. For example, MAPKKs such as AtMKK6 in Arabidopsis and NtMEK1 in tobacco were found directly regulating cytokinesis and mitosis [32,33,34,35]. Although the biological functions of the MAPKs are not fully understood, the MAPKs of same sub-groups are likely to be involved in similar physiological responses [19]. Our previous research has proved that ZjMKK2 was closely related to AtMKK6, and they belong to the same sub-group in the phylogenetic tree [29], so ZjMKK2 might play critical role in the regulation of the physiological processes as AtMKK6 and NtMEK1 did. Meanwhile, our previous study also found that the cytokinin content of Chinese jujube significantly increased after phytoplasma infection; it resulted in the symptom of small leaves and witches’ broom [10]. All of these bioinformatics analyses, physicochemical indexes and apparent symptoms proved that phytoplasma might regulate the transcription of ZjMKK2 gene after inoculation, then cytokinin metabolic pathways of host was modulated, and then morphology of Chinese jujube was altered, such as phyllody, small leaves and witches’ broom. So, ZjMKK2 was selected as the key candidate gene which might play a significant role in jujube-phytolasma interaction.
MAPK cascade genes have been shown to play pivotal roles in regulation of plant defense responses. So far, the best characterized MAPK cascade genes are AtMPK3/6 in Arabidopsis and their orthologs in other plant species. In A. thaliana, AtMPK3/AtMPK6 which belong to Group A have been described to be positive regulators of innate immunity [36,37,38]. In addition, AtMKK7 (belong to Group D) played a critical role in activation of plant systemic acquired resistance in A. thaliana [39]. Our previous study found that ZjMPK2 was homologous to AtMPK3/AtMPK6, which belongs to Group A [29]. Furthermore, ZjMKK4 has close phylogenetic relationship with AtMKK7, and they were classified into Group D [29]. It has been indicated that MAPKs proteins classified in the same groups might serve similar functions in different species [40]. Hence, we can deduce the potential functions of ZjMPK2 and ZjMKK4 according to the known AtMPKs and AtMKKs in Arabidopsis, ZjMPK2 and ZjMKK4 serve as candidate genes might play significant roles in the process of host-phytoplasma interaction. Ye et al. found that four receptor kinase FLS2 genes were down-regulated in phytoplasma-infected jujube, which implied that FLS2/flg22 perception within the pattern-triggered immunity system may exist in the jujube-phytoplasma interaction [30]. In addition, they show the MAPK cascade was activated after phytoplasma infection which could further regulate the induction of WRKY33, this result demonstrated the downstream regulation of MAPK cascade could be WRKY transcription factors. Moreover, MAPK cascade signaling networks were induced by FLS2/flg22 perception in the plant defense to bacterial pathogens [41] and we have demonstrated the protein of ZjMKK2 could interact with ZjMPK2 with two hybrid yeast analysis (Figure S4). Therefore, based on our results and the study by Ye et al. [30], we deduce the following hypothesis of Chinese jujube response to JWB phytoplasma infection (Figure 6). Firstly, the phytoplasma infection could be perceived by FLS2 like receptor, then with the unknown signaling transduction, the ZjMKK2 and ZjMKK4 were induced in different expression pattern between susceptible and resistant cultivars of Chinese jujube, furthermore the ZjMPK2 was activated by ZjMKK2 or ZjMKK4 which could further activate WRKY transcription factor. However, there were maybe different MAPK cascade pathways and regulation modules in susceptible and resistant cultivars of Chinese jujube. ZjMKK2 and ZjMKK4 transcription levels decreased significantly in susceptible cultivar after phytoplasam infection, while their expressions increased strictly in resistant cultivar and in recovered plantlets. The results indicated that phytoplasma infection might activate two ZjMKKs (ZjMKK2 and ZjMKK4), but their functions depended on which downstream MAPK it associates with [30,42]. Subsequently, as the sole candidate MAPK gene, ZjMPK2 expression level significantly increased after phytoplasma infection in most materials. But the transcript abundance of ZjMPK2 in resistant cultivar was down-regulated and was significantly lower than in susceptible cultivar. Meanwhile, ZjMPK2 expression was also significantly down-regulated in recovered plantlets compared to diseased plantlets. These perhaps could serve as evidence for that lower expression level of ZjMPK2 might play vital role in JWB defense response. These differential expression regulations of ZjMPK2, ZjMKK2 and ZjMKK4 genes between susceptible and resistant cultivars may be a resistant mechanism in which plant-phytoplasma interaction are fine-tuned, although additional work is needed to confirm this hypothesis.
Because MAPK cascade kinases are post-translationally regulated by phosphorylation, and their functions depend on the amplitude and kinetics of activation, the loss of a functional gene product might not reveal the exact function of a MAPK cascade. So, a combination of biochemical and genetic studies, including both loss-of-function and gain-of-function approaches, will be required to understand the complex roles of MAPK cascade kinases in plant defense responses.

4. Conclusions

Altogether, our study presents the first gene-family-wide investigation on the systematical expression analysis of MAPK and MAPKK genes in Chinese jujube under phytoplasma infection. Most ZjMPK and ZjMKK genes were responsive to JWB phytoplasma infection. And, ZjMPK2, ZjMKK2 and ZjMKK4 genes were selected as important candidate genes for further functional studies.

5. Materials and Methods

5.1. Diseased, Recovered and Healthy Plantlets

The cultivar for this experiment was Z. jujuba Mill. ‘Goutouzao’. The JWB diseased plantlets (the healthy plantlets were infected by phytoplasma through vitro micrografting [43]) and the recovered plantlets (the JWB diseased plantlets were cultured in antibiotic medium with 25 mg·L−1 tetracycline) were used as test groups, the healthy plantlets were used as control (Figure S1). The JWB phytoplasma concentration in the diseased plantlets, recovered plantlets and healthy plantlets was detected by DAPI staining [44] at histological level (Figure S2). Every ten plantlets were pooled as one sample, three independent biological replications were sampled separately, then immediately frozen in liquid nitrogen and stored at −80 °C until RNA extraction.

5.2. The Tissues Showing Different JWB Disease Symptoms

The cultivar for this experiment was Z. jujuba Mill. ‘Dongzao’. Small leaves (shoot with witches-broom), phyllody (floral organs becoming leaf-like) and non-symptomatic leaves were used as test group which selected from the same branch of diseased tree, and the normal leaves from healthy trees were used as control, as shown in Figure S5. The detection of JWB phytoplasmas in the infected materials in June was conducted by DAPI staining at histological level and quantitative realtime PCR analysis (qRT-PCR) at the molecular level from June to October [44]. All samples were collected at five stages (on the 15th day of each month from June to October). Each material was sampled with three replicates from independent trees. All samples were rapidly frozen in liquid nitrogen and kept at −80 °C for RNA isolation.

5.3. Resistant and Susceptible Cultivars

A JWB-resistant cultivar (Z. jujuba Mill. ‘Xingguang’) and a JWB-susceptible cultivar (Z. jujuba Mill. ‘Junzao’) were used as scions for top grafting (as shown in Figure S6). The rootstock was JWB-susceptible cultivar Z. jujuba Mill. ‘Dongzao’. The ‘Xingguang’ and ‘Junzao’ scions were grafted on rootstocks with JWB (Test group) and healthy ones (Control group), respectively. Three replicates were conducted of each grafting treatment. All the experimental trees were cultivated in the natural environmental conditions. The mature leaves in the middle of bearing shoot from sprouted scions were collected at five stages (on the 15th day of each month from June to October). The sampled leaves were frozen by liquid nitrogen immediately and stored at −80 °C until RNA extraction.
‘Xingguang’ (resistant cultivar) and ‘Junzao’ (susceptible cultivar) sprouted around 25 days after grafting on rootstocks (May 15th). The resistant cultivar ‘Xingguang’ only displayed slight symptoms at the initial stage after grafting (60 days, June 20th) and then reverted to normal growth. In infected ‘Junzao’, the visual JWB symptoms, such as elongated pedicel and virescent flowers, were firstly detected at 45 days after grafting (June 5th), and then small leaves and witches’ broom were observed at 60 days after grafting inoculation (June 20th). The JWB phytoplasma presence of the samples was detected by quantitative real-time PCR (qRT-PCR) [44]. The expression of phytoplasma TMK gene in jujube samples was analyzed and ZjACT was used as an internal control. The detection of the phytoplasma in the two cultivars was shown in Figure S7.

5.4. Total RNA Extraction

Total RNA of the samples was isolated according to the manufacturer’s instructions of TIANGEN RNA Extraction Kit. DNaseⅠ treatment was used to remove contaminating genomic DNA. TaKaRa RNA PCR Kit (AMV) Ver.3.0 (TaKaRa, Dalian, China) was applied in the synthesis of double-stranded cDNA according to the instructions.

5.5. Quantitative Real-Time PCR System

The Quantitative Real-time PCR (qRT-PCR) was carried out on the Bio-Rad iQ™ 5 using TransStart Top Green qPCR SuperMix AQ131 (TransGen Biotech, Beijing, China). The 20 µL reaction system contained 10 µL of 2×SYBR Premix ExTaq™ (TransGen Biotech, Beijing, China), 0.4µL each of 10 µM primers, 1µL diluted cDNA and 8.2 µL ddH2O. The thermal profile was pre-incubation for 3 min at 94 °C, followed by 40 cycles of 5 s at 94 °C, 15 s at 55~63 °C and 15 s at 72 °C.

5.6. Expression Analysis

The expression profile analysis of the target genes was carried out by qRT-PCR, with ZjActin as the internal control [45]. Primer sequences for qRT-PCR analysis were shown in our previous study [29]. The relative expression levels were calculated by the 2−∆∆CT method [46]. The gene’s relative expression change more than two-fold was noted as significant difference and the gene was considered as differentially expressed genes (DEGs) [30].

5.7. Heatmap Construction

The expression profiles of all ZjMPK and ZjMKK genes in different samples were illustrated by a color gradient heatmap. The heatmap was constructed by heatmap software Heml 1.0 using expression fold-changes.

5.8. Protein-Protein Interaction Analysis

To determine the interaction network of candidate genes, STRING database [47] was used to predict protein-protein interactions. Firstly, the nucleotide sequences of candidate genes were translated into amino acid sequences by Primer Premier 5. Secondly, Arabidopsis thaliana was selected as reference template. Lastly, the amino acid sequences of candidate genes were used as query sequences to search interaction pathway in STRING database.

5.9. Yeast Two-Hybrid Screening (Y2H)

ZjMKK2 fused to the GAL4 activation domain (AD) was expressed in combination with ZjMPK2 fused to the GAL4 DNA-binding domain (BD) in yeast strain AH109. Then the yeast cells were spotted on selective medium lacking leucine/tryptophan (-LW), lacking leucine/tryptophan/histidine (-LWH) and lacking tryptophan/leucine/adenine/histidine (-LWAH), respectively. The BD-fused ZjMPK2 was co-expressed with empty AD as the negative control. The plates were incubated for 3 days at 30 °C.

5.10. Statistical Analysis

Data were shown as the means ± standard deviation (SD) of independent experiments. Statistical analysis was performed by SPSS 16.0 software (IBM, Armonk, NY, USA) using Independent-Samples T-test and One-Way ANOVA method.

Supplementary Materials

The following are available online at https://www.mdpi.com/1999-4907/10/5/392/s1, Figure S1: The healthy, diseased, and recovered plantlets. A: Healthy plantlets; B: Diseased plantlets; C: Recovered plantlets. Bar = 2 cm. Figure S2: Phytoplasma determination in jujube petiole phloem by using 4’,6-diamidino-2-phenylindole (DAPI). A, No fluorescent spots in the sieve element (SE) of healthy plantlets. B, The fluorescent spots formed a large bright circle in the SE of the diseased plantlets. C, No fluorescent spots in the SE of recovered plantlets. The number and size of fluorescent spots represent the number of phytoplasma. Bar = 100 µm. Figure S3: The protein-protein interaction analysis of ZjMPK2, ZjMKK2 and ZjMKK4 by STRING database. Figure S4: Interaction between ZjMPK2 and ZjMPKK2 with yeast two-hybrid analysis. ZjMKK2 fused to the GAL4 activation domain (AD) was expressed in combination with ZjMPK2 fused to the GAL4 DNA-binding domain (BD) in yeast strain AH109. Then the yeast cells were spotted on selective medium lacking leucine/tryptophan (-LW), lacking leucine/tryptophan/histidine (-LWH) and lacking tryptophan/leucine/adenine/histidine (-LWAH), respectively. The BD-fused ZjMPK2 was co-expressed with empty AD as the negative control. Figure S5: The tissues showing different JWB disease symptoms. A: Small leaves; B: Phyllody; C: Non-symptomatic leaves; D: Healthy leaves. Bar = 2 cm. A, B and C were used as test group which collected from the diseased trees. D was used as control which collected from the healthy trees. Figure S6: The resistant and susceptible cultivars were grafted on JWB diseased rootstocks. The JWB symptoms of small leaves, phyllody and witches’-broom in susceptible cultivar were observed, but the resistant cultivar scions only showed symptom slightly at initial stage after grafting inoculation and then reversed to normal growth. Figure S7: Expression analysis of phytoplasma TMK in susceptible and resistant varieties which were grafted on JWB diseased rootstocks. Table S1: The number of ZjMPK and ZjMKK genes with significant difference expression in JWB-resistant/susceptible cultivars at five stages after phytoplasma infection.

Author Contributions

Data curation, Z.L., Z.Z., L.W. (Lili Wang), C.F., L.Z. and Z.Y.; Formal analysis, Z.L. and Z.Z.; Funding acquisition, Z.L., J.Z. and M.L.; Investigation, C.X. and L.W. (Lixin Wang); Project administration, Z.L., J.Z. and M.L.; Resources, C.X. and L.W. (Lixin Wang); Supervision, J.Z. and M.L.; Writing – original draft, Z.L. and Z.Z.; Writing – review & editing, J.Z. and M.L.

Acknowledgments

This work was supported by the Funds for Youth Fund of Hebei Province Natural Science Foundation (grant number C2016204157); Hebei Distinguished Young Scholar (grant number C2016204145); Significance Fund of Hebei Province Natural Science Foundation (grant number C2017204114); National Science and Technology Support Plan of China (grant number 2013BAD14B03); Agricultural University of Hebei Foundation for Leaders of Disciplines in Science Technology.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. The expression modules of ZjMPK and ZjMKK genes in diseased plantlets and recovered plantlets. ZjACT was used as the internal standard. The healthy plantlets were used as control. The mean expression value was calculated from 3 independent replicates. The vertical bars indicate the standard deviation. Different letters indicate significant difference at the level of p < 0.05.
Figure 1. The expression modules of ZjMPK and ZjMKK genes in diseased plantlets and recovered plantlets. ZjACT was used as the internal standard. The healthy plantlets were used as control. The mean expression value was calculated from 3 independent replicates. The vertical bars indicate the standard deviation. Different letters indicate significant difference at the level of p < 0.05.
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Figure 2. The expression profiles of ZjMPK and ZjMKK genes in tissues showing different JWB disease symptoms. The heatmap was generated by Heml 1.0 using expression fold-change. Up-regulated genes were colored in red, down-regulated genes were colored in blue (color code in the bottom).
Figure 2. The expression profiles of ZjMPK and ZjMKK genes in tissues showing different JWB disease symptoms. The heatmap was generated by Heml 1.0 using expression fold-change. Up-regulated genes were colored in red, down-regulated genes were colored in blue (color code in the bottom).
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Figure 3. The expression trends of ZjMPK2 and ZjMKK2 genes in tissues showing different JWB disease symptoms. ZjACT was used as the internal standard. The leaves from healthy trees were used as control. The mean expression value was calculated from 3 independent replicates. The vertical bars indicate the standard deviation.
Figure 3. The expression trends of ZjMPK2 and ZjMKK2 genes in tissues showing different JWB disease symptoms. ZjACT was used as the internal standard. The leaves from healthy trees were used as control. The mean expression value was calculated from 3 independent replicates. The vertical bars indicate the standard deviation.
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Figure 4. The expression profiles of ZjMPK and ZjMKK genes in JWB-resistant/susceptible cultivars after phytoplasma infection. The heatmap was generated by Heml 1.0 using expression fold-change. Up-regulated genes were colored in red, down-regulated genes were colored in blue (color code in the bottom).
Figure 4. The expression profiles of ZjMPK and ZjMKK genes in JWB-resistant/susceptible cultivars after phytoplasma infection. The heatmap was generated by Heml 1.0 using expression fold-change. Up-regulated genes were colored in red, down-regulated genes were colored in blue (color code in the bottom).
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Figure 5. The expression modules of ZjMPK2, ZjMKK2 and ZjMKK4 genes in JWB-resistant/susceptible cultivars after phytoplasma infection. ZjACT was used as the internal standard. The ‘Xingguang’ and ‘Junzao’ scions which grafted on healthy rootstocks were used as control. The mean expression value was calculated from 3 independent replicates. The vertical bars indicate the standard deviation. p < 0.05 and p < 0.01 were considered as significant (shown as *) and highly significant difference (shown as **), respectively.
Figure 5. The expression modules of ZjMPK2, ZjMKK2 and ZjMKK4 genes in JWB-resistant/susceptible cultivars after phytoplasma infection. ZjACT was used as the internal standard. The ‘Xingguang’ and ‘Junzao’ scions which grafted on healthy rootstocks were used as control. The mean expression value was calculated from 3 independent replicates. The vertical bars indicate the standard deviation. p < 0.05 and p < 0.01 were considered as significant (shown as *) and highly significant difference (shown as **), respectively.
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Figure 6. Possible MAPK cascades pathway of Chinese jujube under JWB phytoplasma infection. Different color arrows indicate up-regulation (Red) and down-regulation (Blue).
Figure 6. Possible MAPK cascades pathway of Chinese jujube under JWB phytoplasma infection. Different color arrows indicate up-regulation (Red) and down-regulation (Blue).
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Table
Table 1. The number of ZjMPK and ZjMKK genes with significant difference expression in non-symptomatic leaves, phyllody and small leaves at five stages.
Table 1. The number of ZjMPK and ZjMKK genes with significant difference expression in non-symptomatic leaves, phyllody and small leaves at five stages.
Gene NameUp-Regulated Expression TimesDown-Regulated Expression TimesTotal Times
ZjMPK1011
ZjMPK29110
ZjMPK3044
ZjMPK4022
ZjMPK5224
ZjMPK6022
ZjMPK7044
ZjMPK8000
ZjMPK9707
ZjMPK10011
ZjMKK1000
ZjMKK2369
ZjMKK3000
ZjMKK4011
In all212445
Note: The gene’s relative expression changes more than two-fold were considered as significant differences. The significant difference times of one gene is the sum of that gene with significant difference expression in non-symptomatic leaves, phyllody and small leaves at five stages, which include Up-regulated expression times and Down-regulated expression times. Up-regulated expression (Down-regulated expression) means that the gene’s relative expression level higher (lower) than 2 fold-changes.

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Liu, Z.; Zhao, Z.; Xue, C.; Wang, L.; Wang, L.; Feng, C.; Zhang, L.; Yu, Z.; Zhao, J.; Liu, M. Three Main Genes in the MAPK Cascade Involved in the Chinese Jujube-Phytoplasma Interaction. Forests 2019, 10, 392. https://doi.org/10.3390/f10050392

AMA Style

Liu Z, Zhao Z, Xue C, Wang L, Wang L, Feng C, Zhang L, Yu Z, Zhao J, Liu M. Three Main Genes in the MAPK Cascade Involved in the Chinese Jujube-Phytoplasma Interaction. Forests. 2019; 10(5):392. https://doi.org/10.3390/f10050392

Chicago/Turabian Style

Liu, Zhiguo, Zhihui Zhao, Chaoling Xue, Lixin Wang, Lili Wang, Chunfang Feng, Liman Zhang, Zhe Yu, Jin Zhao, and Mengjun Liu. 2019. "Three Main Genes in the MAPK Cascade Involved in the Chinese Jujube-Phytoplasma Interaction" Forests 10, no. 5: 392. https://doi.org/10.3390/f10050392

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