Genome-wide identification of MAPKKK genes and their responses to phytoplasma infection in Chinese jujube (Ziziphus jujuba Mill.).

BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades play vital roles in signal transduction in response to a wide range of biotic and abiotic stresses. In a previous study, we identified ten ZjMAPKs and five ZjMAPKKs in the Chinese jujube genome. We found that some members of ZjMAPKs and ZjMAPKKs may play key roles in the plant's response to phytoplasma infection. However, how these ZjMAPKKs are modulated by ZjMAPKKKs during the response process has not been elucidated. Little information is available regarding MAPKKKs in Chinese jujube. RESULTS: A total of 56 ZjMAPKKKs were identified in the jujube genome. All of these kinases contain the key S-TKc (serine/threonine protein kinase) domain, which is distributed among all 12 chromosomes. Phylogenetic analyses show that these ZjMAPKKKs can be classified into two subfamilies. Specifically, 41 ZjMAPKKKs belong to the Raf subfamily, and 15 belong to the MEKK subfamily. In addition, the ZjMAPKKKs in each subfamily share the same conserved motifs and gene structures. Only one pair of ZjMAPKKKs (15/16, on chromosome 5) was found to be tandemly duplicated. Using qPCR, the expression profiles of these MAPKKKs were investigated in response to infection with phytoplasma. In the three main infected tissues (witches' broom leaves, phyllody leaves, and apparently normal leaves), ZjMAPKKK26 and - 45 were significantly upregulated, and ZjMAPKKK3, - 43 and - 50 were significantly downregulated. ZjMAPKKK4, - 10, - 25 and - 44 were significantly and highly induced in sterile cultivated tissues infected by phytoplasma, while ZjMAPKKK6, - 7, - 17, - 18, - 30, - 34, - 35, - 37, - 40, - 41, - 43, - 46, - 52 and - 53 were significantly downregulated. CONCLUSIONS: For the first time, we present an identification and classification analysis of ZjMAPKKKs. Some ZjMAPKKK genes may play key roles in the response to phytoplasma infection. This study provides an initial understanding of the mechanisms through which ZjMAPKKKs are involved in the response of Chinese jujube to phytoplasma infection.

Defense Responses, Induced by p-Coumaric Acid and Methyl p-Coumarate, of Jujube ( Ziziphus jujuba Mill.) Fruit against Black Spot Rot Caused by Alternaria alternata.

The esterified fraction of jujube ( Ziziphus jujuba Mill.) peel extract showed strong antifungal activity on Alternaria alternata. p-Coumaric acid (pCA) was found to be the most predominant individual phenolic acid that was correlated highly with the antifungal activity of the esterified fraction. Thus, effects of postharvest treatments with pCA and its simplest esterified derivative methyl p-coumarate (MeCA) against black spot rot on jujube fruit caused by A. alternata were investigated. pCA and MeCA strongly suppressed in vitro growth of the fungus and significantly reduced postharvest Alternaria rot on fresh jujubes. Biochemical and transcriptional analysis revealed that pCA and MeCA regulated the expression of some genes encoding antioxidant enzymes and their enzymatic activities, enhanced the phenylpropanoid pathway metabolism, and activated the expression of genes encoding pathogenesis-related proteins. These results suggested that, apart from its direct antifungal activity, pCA and MeCA induced defense responses in jujube fruit against postharvest Alternaria rot.

Changing Host Photosynthetic, Carbohydrate, and Energy Metabolisms Play Important Roles in Phytoplasma Infection.

Phytoplasmas parasitize plant phloem tissue and cause many economically important plant diseases. Jujube witches'-broom disease is a destructive phytoplasma disease of Chinese jujube (Ziziphus jujuba). To elucidate the influence of phytoplasma on host photosynthetic, carbohydrate and energy metabolisms, four types of jujube tissues showing disease symptoms with different severity were investigated at the structural, physiological, and molecular levels. Quantitative real-time PCR and high-performance liquid chromatography results showed that the down-regulation of genes related to photosynthesis and the lower contents of chlorophyll in diseased leaves. This clearly inhibited the light-harvesting and photosystem II activity of photosynthesis; however, overexpression of genes related to starch, sucrose and glucose synthesis led to higher contents of these carbohydrates. Meanwhile, transmission electron microscopy images revealed that dense amounts of phytoplasmas accumulated in the sieve elements of diseased petiole phloem, and the structure of the grana and stroma lamellae of chloroplasts in the diseased leaves was destroyed. Phytoplasma infection inhibited photosynthesis and led to abnormal carbohydrate accumulation in the diseased leaves. Furthermore, comparative metabolite analysis indicated that phytoplasma infection also stimulated amino acids and energy metabolisms of the diseased leaves. Continually inhibiting the photosynthetic process and stimulating carbohydrate and energy metabolisms of diseased trees may exhaust their nutrients. Our results highlight the importance of changing host metabolisms during the pathogenic process.

Plant chitinase III Ziz m 1 stimulates multiple cytokines, most predominantly interleukin-13, from peripheral blood mononuclear cells of latex-fruit allergic patients.

BACKGROUND: Indian jujube is a fruit abundantly cultivated in Taiwan. Its major allergen in latex-fruit syndrome is Ziz m 1 of the chitinase III family. The Ziz m 1 Pichia (rZiz m 1-P) has chitinase activity but not Ziz m 1 E. coli (rZiz m 1-E). OBJECTIVE: This study examined whether plant chitinase III, using rZiz m 1-P and rZiz m 1-E, can stimulate allergic inflammation similar to that of mammalian chitinases. METHODS: Five patients allergic to latex-Indian jujube and five nonallergic controls were evaluated. Their peripheral blood mononuclear cells (PBMC) were cultured with rZiz m 1-E or rZiz m 1-P and pulsed with phorbol 12-myristate 13-acetate. Eleven cytokines were measured by FlowCytomix human Th1/Th2 plex kit and interleukin (IL)-13 by sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: Interleukin-13 significantly increased in rZiz m 1-P stimulated PBMC of allergic subjects but was undetectable when stimulated with rZiz m 1-E. The stimulation index significantly increased in IL-13 (380.6 ± 77.33 vs 13.70 ± 6.92), IL-5 (6.70 ± 0.59 vs 0.70 ± 0.37), IL-1ß (32.70 ± 0.83 vs 2.10 ± 1.29), and tumor necrosis factor beta (TNF-ß) (17.10 ± 2.66 vs 1.50 ± 0.66) between allergic and nonallergic subjects after rZiz m 1-P stimulation. There was no difference in terms of IL-2, IFN-γ, IL-8, and TNF-α production. CONCLUSIONS: The biological function of chitinase activity is required for Ziz m 1 to induce a Th2-specific immune response. This is the first report on PBMC responses of latex-fruit syndrome subjects toward an active exogenous plant class III chitinase that can stimulate multiple cytokines, especially IL-13, from allergic subjects. This implies the role of cross-reactive food allergens in propagating allergic inflammation among allergic subjects.

Augmentation of expression of immunocytes' functions by seed extract of Ziziphus mauritiana (Lamk.).

AIM OF THE STUDY: The present study was carried out to evaluate the immunomodulatory potential of Ziziphus mauritiana (Lamk.) seed extract to ascertain the folkloric claim as immunomodulator. MATERIALS AND METHODS: The aqueous-ethanolic seed extract (100-400 mg kg(-1)) of Z. mauritiana was investigated for immunomodulatory potential in mice. The extract was standardized with HPLC using betulinic acid as a marker. Functions of various immunocytes in the form of humoral (development of anti-SRBC (sheep red blood cells) antibody titers) and cell-mediated immune response (delayed type hypersensitivity, nitroblue tetrazolium reduction, inducible nitric oxide synthase activity and bactericidal activity) was studied in SRBC immunized mice. The cytokine, IFN-gamma (interferon-gamma) and IL-4 (interleukin-4) secretion was also measured quantitatively by ELISA as the expression of functions of Th-1 and Th-2 respectively. Levamisole (2.5 mg kg(-1)) was used as standard drug. RESULTS: The seed extract demonstrated significant (P<0.05-0.001) up-regulation of cell-mediated, humoral immune response and Th-1 mediated cytokine IFN-gamma and decline in Th-2 mediated cytokine IL-4. At higher dose of extract the results were comparable to that of the levamisole. CONCLUSION: The immunostimulatory potential of this seed extract is likely to be mediated through its effect on macrophage function and Th-1 mediated immunity confirming the folkloric use of this plant.

Identification of immunoglobulin E (IgE)-binding epitopes and recombinant IgE reactivities of a latex cross-reacting Indian jujube Ziz m 1 allergen.

Ziz m 1 is a major Indian jujube (Zizyphus mauritiana) allergen involved in latex-fruit syndrome, and cDNA of the allergen has been cloned, sequenced and expressed in yeast by our laboratory previously. In this study, we performed an immunoglobulin E (IgE)-binding epitope analysis of Ziz m 1 using overlapping recombinant fragments. Eight overlapping recombinant fragments were generated from the recombinant Ziz m 1 allergen. The fragments were expressed in Escherichia coli and IgE-binding activities were evaluated by sera of latex-Indian jujube-allergic subjects and normal subjects using immunoblotting. Human allergic sera are not able to recognize fragments consisting of amino acid sequences 26-71, 119-280 and 119-291. However, residues at positions 26-199, 26-105, 26-86, 119-320 and 238-330 were found relevant in the IgE-binding. Our results indicate that (72)NISGHCSDCTFLGEE(86) and (292)VWNRYYDLKTNYSSSIILEYVNSGTKYLP(320) of Ziz m 1 are the sequences required for human IgE binding. Four corresponding peptides, (72)NISGHCSDCTE(86), (292)VWNRYYDLKT(301), (300)KTNYSSSIILEY(311) and (309)LEYVNSGTKYLP(320), were synthesized, and these peptides reacted with 70%, 100%, 70% and 70% of 10 allergic sera tested, as revealed by enzyme-linked immunosorbent assay. Sensitization to (292)VWNRYYDLKT(301) correlated significantly with the presence of allergic symptoms (P < 0.001). These findings will be useful in designing diagnostic and therapeutic approaches, thereby contributing to the development of specific immunotherapy for subjects with latex-fruit syndrome.

Molecular cloning of Indian jujube (Zizyphus mauritiana) allergen Ziz m 1 with sequence similarity to plant class III chitinases.

Indian jujube (Zizyphus mauritiana) is a sweet fruit that is abundantly cultivated in Taiwan. We have previously identified 42 and 30 kDa allergens that are cross-reactive with latex allergen from crude Indian jujube extract. This study aimed to clone the 30 kDa Ziz m 1 Z. mauritiana allergen. The Ziz m 1 encoding cDNA was isolated from a ZAPII cDNA library constructed from Z. mauritiana mRNA, sequenced and expressed in Pichia pastoris. The protein predicted from the cDNA sequence has 330 amino acids, the first 25 of which constituted a putative signal peptide. The deduced molecular mass of the mature protein is 33.86 kDa, while its isoelectric point is estimated at 4.36. The recombinant Ziz m 1 showed chitinase activity, possessed IgE binding capacity, and had IgE cross-reactivity with the latex allergen. Moreover, anti-recombinant Ziz m 1 antibody-based ELISA was able to detect commercial skin testing latex reagent, laboratory prepared latex and Indian jujube extracts. Recombinant Ziz m 1 showed 87.5% skin reactivities on eight latex- and Indian jujube-sensitive subjects. Although no sequence similarity was found to other known allergens, Ziz m 1 was found to have amino acid sequence identity (39-45.3%) to many plant chitinases including chitinase (45.2%) of Hevea brasiliensis (hevamine), class III chitinases of Vigna angularis (45.3%), Capsicum annuum (44.7%) and Oryza sativa (41.2%). A conserved domain search revealed that Ziz m 1 belongs to the family 18 glycosyl hydrolases. The recombinant allergen may therefore be of value for diagnosis and therapeutic purposes, and the further characterization of Indian jujube allergen may help to elucidate the mechanism underlying latex-fruit syndrome.

Allergenic components of Indian jujube (Zizyphus mauritiana) show IgE cross-reactivity with latex allergen.

BACKGROUND: 'Latex-fruit syndrome' has been well documented. A prevalence of latex allergy among medical workers of 6.8-8.6% had been reported in Taiwan. However, there has been no study to determine the importance and type of fruit hypersensitivity in latex-allergic patients in Taiwan. This study aimed to identify the allergenic components of Indian jujube (Zizyphus mauritiana) and characterize the cross-reactivity of specific IgE antibodies to latex allergen. METHODS: Crude extracts were prepared from Indian jujube and from ammoniated natural rubber latex, and six medical workers and one patient with a history of fruit allergy underwent skin testing with routine allergens, latex, Indian jujube and other fruits. Sera from two Indian jujube skin test-positive latex-allergic subjects were used for allergen-specific IgE, immunoblotting, immunoblot inhibition and enzyme-linked immunosorbent assay (ELISA) inhibition studies. RESULTS: Both patients had positive skin test responses and specific IgE assays to Indian jujube and latex extracts. Immunoblotting revealed that IgE from both subjects bound to a 42-kD latex protein and a 42-kD Indian jujube protein. In addition, IgE from one subject bound to a 30-kD Indian jujube protein. Preincubation of atopic sera with Indian jujube or latex extract demonstrated absent and/or marked inhibition of IgE binding. Moreover, anti-Indian jujube protein antibody-based ELISA was able to detect latex extracts. CONCLUSIONS: Our results add to findings regarding the 'latex-fruit syndrome' described in the literature, and further study of the cross-reacting allergens identified in Indian jujube may help to elucidate the mechanisms underlying this syndrome.