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. 2018 Sep;17(9):1702-1719.
doi: 10.1074/mcp.RA118.000670. Epub 2018 May 30.

Integrated Phloem Sap mRNA and Protein Expression Analysis Reveals Phytoplasma-infection Responses in Mulberry

Ying-Ping Gai  1 , Shuo-Shuo Yuan  2 , Zhao-Yang Liu  2 , Huai-Ning Zhao  1 , Qi Liu  1 , Rong-Li Qin  1 , Li-Jing Fang  2 , Xian-Ling Ji  3
Affiliations

Integrated Phloem Sap mRNA and Protein Expression Analysis Reveals Phytoplasma-infection Responses in Mulberry

Ying-Ping Gai et al. Mol Cell Proteomics. 2018 Sep.

Abstract

To gain insight into the response of mulberry to phytoplasma-infection, the expression profiles of mRNAs and proteins in mulberry phloem sap were examined. A total of 955 unigenes and 136 proteins were found to be differentially expressed between the healthy and infected phloem sap. These differentially expressed mRNAs and proteins are involved in signaling, hormone metabolism, stress responses, etc. Interestingly, we found that both the mRNA and protein levels of the major latex protein-like 329 (MuMLPL329) gene were increased in the infected phloem saps. Expression of the MuMLPL329 gene was induced by pathogen inoculation and was responsive to jasmonic acid. Ectopic expression of MuMLPL329 in Arabidopsis enhances transgenic plant resistance to Botrytis cinerea, Pseudomonas syringae pv tomato DC3000 (Pst. DC3000) and phytoplasma. Further analysis revealed that MuMLPL329 can enhance the expression of some defense genes and might be involved in altering flavonoid content resulting in increased resistance of plants to pathogen infection. Finally, the roles of the differentially expressed mRNAs and proteins and the potential molecular mechanisms of their changes were discussed. It was likely that the phytoplasma-responsive mRNAs and proteins in the phloem saps were involved in multiple pathways of mulberry responses to phytoplasma-infection, and their changes may be partially responsible for some symptoms in the phytoplasma infected plants.

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Conflict of interest statement

Conflict of interest: We declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Western blotting and Northern blotting analyses for the presence of Rubisco and CmPP16. A, Total proteins extracted from mulberry leaf tissue free of major veins and phloem sap were analyzed for the presence of Rubisco by Western blotting. B and C, Total RNAs extracted from mulberry leaf tissue free of major veins and phloem sap were analyzed for the presence of Rubisco (B) and CmPP16 RNAs (C) by Northern blotting. IPS, phloem sap sampled from infected plants; HSP phloem sap sampled from healthy plants. LT, leaf tissue free of major veins.
Fig. 2.
Fig. 2.
Expression abundance analysis of the unigenes by real-time PCR. The relative abundance of the unigenes was evaluated using the comparative Ct method with EF1-α as the reference gene. The log2 values of the ratio of phytoplasma-infected samples to the healthy samples are plotted. Values are given as the mean ± standard deviation (S.D.) of three experiments in each group.
Fig. 3.
Fig. 3.
MapMan overview of differentially regulated genes involved in different metabolic processes (A), signaling pathways (B) and stress responses (C) in infected phloem sap and healthy phloem saps. Individual genes are represented by small squares. Blue denotes upregulation and red downregulation.
Fig. 4.
Fig. 4.
Distribution percentage of the differentially expressed proteins in various categories.
Fig. 5.
Fig. 5.
Northern blotting and Western blotting analyses of MuMLPL329 gene expression in phytoplasma-infected and healthy phloem saps. A, Northern blot analysis of MuMLPL329 expression in phytoplasma-infected and healthy phloem saps. Total RNA (10 μg) was loaded into each lane, and the DIG-labeled RNA probes for MuMLPL329 gene were used. B. Western blotting analyses of MuMLPL329 expression in phytoplasma-infected and healthy phloem saps. Whole soluble proteins were sampled from phytoplasma-infected and healthy plant phloem saps and were separated by 12% SDS-PAGE. The gels was transferred onto a PVDF membrane and incubated with the polyclonal antiserum produced against the MuMLPL329 protein.
Fig. 6.
Fig. 6.
Multiple sequence alignment between MuMLPL329 and other MLP proteins. Amino acid residues conserved in all proteins were black shaded and similar amino acids were red or blue shaded. The aligned sequences included those MLP-like protein 328 from Arabidopsis (NP 565264.1), MLP-like protein 329 from Arabidopsis (NP 565265.1), MLP-like protein 329 from Prunus persica (XP 007225991.1), MLP-like protein 328 from Malus domestica (XP 008389200.1), MLP-like protein 28 from Malus domestica (XP 008389201.1), MLP-like protein 329 from M. notabilis (XP 010086735.1), MLP-like protein 328 from M. notabilis (XP 010086736.1), MLP-like protein 28 from Juglans regia (XP 018824604.1), MLP-like protein 328 from Manihot esculenta (XP 021593664.1). MLP-like protein 328 from Prunus avium (XP 021823275.1), and MLP-like protein 329 from Cucurbita moschata (XP 022942726.1).
Fig. 7.
Fig. 7.
Proposed 3-D structures of the MuMLPL329 proteins established by homology-based modeling and phylogenetic relationship between MuMLPL329 and MLPs in other plants. A. Three-dimensional model of the MuMLPL329 protein was constructed by SWISS-MODEL. The Y-shaped hydrophobic cavity is formed by a seven-stranded-sheet wrapped around a long C-terminal helix, and is closed at one end by two short helices. B, Phylogenetic analysis was performed using the neighbor-joining method. The scale indicates branch lengths. Bootstrap values below 80 are indicative of low confidence. The accession numbers of the proteins are indicated.
Fig. 8.
Fig. 8.
Subcellular localization of MuMLPL329 fused with EGFP. MuMLP-L329::EGFP vector was transiently coexpressed in Arabidopsis mesophyll protoplasts and visualized with a confocal laser scanning microscope (Zeiss LSM880, Zeiss, Jena, Germany) after 12 h. Fluorescence was detected using a 493 to 550-nm band-pass filter for GFP and a 660 to 700 nm band-pass filter for chloroplast auto-fluorescence. Image processing was performed with the Zeiss LSM image processing software (Zeiss).
Fig. 9.
Fig. 9.
Expression pattern of the MuMLPL329 gene in various organs of mulberry plant (A) and expression of MuMLPL329 in leaves after treatments with JA, SA, P. syringae pv. mori or C. dematium, respectively (B). Gene expression was determined by qRT-PCR in different tissues. EF1-α was used as the reference gene. Data represent the mean ± S.D. of three independent biological samples. Different letters above the columns indicate significant differences (p < 0.05) according to Duncan's multiple range tests. Ps: P. syringae pv. mori. Cd: C. dematium.
Fig. 10.
Fig. 10.
Transient expression of the pMuMLPL329::GUS fusion in Nicotiana benthamiana leaves. Tobacco leaves were infiltrated with transformed Agrobacterium and then were treated with Pst. DC3000 or B. cinerea. Leaves were sampled at 36 h and 72 h after Pst. DC3000 or B. cinerea treatment, respectively. For SA and JA treatments, leaves were sampled at 6 h after treatment.
Fig. 11.
Fig. 11.
Enhanced disease tolerance of the Arabidopsis plants overexpressing MulMLPL329. A, Resistance analysis of the transgenic Arabidopsis plants to Pst. DC3000; B, Growth of Pst. DC3000 strains within infected Arabidopsis leaves; C, Resistance analysis of the transgenic Arabidopsis plants to B. cinerea. The bacterial numbers were calculated at 12-h intervals after inoculation and represented as CFU per gram of leaf tissue. The data represent the means and S.D. of three biological samples. Double asterisks indicate significant differences versus WT (p < 0.01). WT. Wild-type Arabidopsis; OE1, OE2 and OE3: Transgenic Arabidopsis lines overexpressing MulMLPL329.
Fig. 12.
Fig. 12.
Phenotypes of the phytoplasma-infected Arabidopsis and detection of phytoplasma. A, Phenotypes of phytoplasma-infected MuMLPL329 transgenic Arabidopsis. B, Phenotypes of phytoplasma-infected wild type Arabidopsis. C, qRT-PCR amplification of the 16S rDNA of phytoplasma to detect phytoplasma. Error bars indicate the S.D. of three technical replicates within one biological experiment. Three biological repeats were performed.
Fig. 13.
Fig. 13.
Expression of defense-related genes in MuMLPL329-overexpressing plants. Error bars indicate the S.D. of three technical replicates within one biological experiment. Three biological repeats were performed. OE1, OE2 and OE3: Transgenic Arabidopsis lines overexpressing MulMLPL329.
Fig. 14.
Fig. 14.
Total flavonoid content of mulberry and Arabidopsis leaves. Data represent the mean ± S.D. of three independent biological samples. Different letters above the columns indicate significant differences (p < 0.05) according to Duncan's multiple range tests. Three biological repeats were performed. OE1, OE2 and OE3: Transgenic Arabidopsis lines overexpressing MulMLPL329.
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