CmMYB19 Over-Expression Improves Aphid Tolerance in Chrysanthemum by Promoting Lignin Synthesis

Abstract

The gene encoding the MYB (v-myb avian myeloblastosis vira l oncogene homolog) transcription factor CmMYB19 was isolated from chrysanthemum. It encodes a 200 amino acid protein and belongs to the R2R3-MYB subfamily. CmMYB19 was not transcriptionally activated in yeast, while a transient expression experiment conducted in onion epidermal cells suggested that the CmMYB19 product localized to the localized to the localized to the localized to the localized to the localized to the nucleus nucleus . CmMYB19 transcription was induced by aphid (Macrosiphoniella sanborni) infestation, and the abundance of transcript was higher in the leaf and stem than in the root. The over-expression of CmMYB19 restricted the multiplication of the aphids. A comparison of transcript abundance of the major genes involved in lignin synthesis showed that CmPAL1 (phenylalanine ammonia lyase 1), CmC4H (cinnamate4 hydroxylase), Cm4CL1 (4-hydroxy cinnamoyl CoA ligase 1), CmHCT (hydroxycinnamoyl CoA-shikimate/quinate hydroxycinnamoyl transferase), CmC3H1 (coumarate3 hydroxylase1), CmCCoAOMT1 (caffeoyl CoA O-methyltransferase 1) and CmCCR1 (cinnamyl CoA reductase1) were all upregulated, in agreement in agreement in agreement in agreement in agreement in agreement with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content in CmMYB19 over-expressing plants plants plants. Collectively, the over-expression of CmMYB19 restricted the multiplication of the aphids on the host, mediated by an enhanced accumulation of lignin.

Keywords: Chrysanthemum morifolium; aphid; expression analysis; lignin; transgenic plants.

Figure 1

Alignment of the putative CmMYB19 protein sequence with those of R2R3 MYBs from lignin biosynthesis or wall deposition group in Arabidopsis. The two MYB domains are indicated by lines drawn below the alignment.

Figure 2

Phylogenetic analysis of CmMYB19 (boxed) and A. thaliana R2R3-MYB members. The alignment was generated by ClustalW (available on: http://www.ebi.ac.uk/Tools/msa/ clustalw2/), and the neighbor-joining tree constructed using MEGA 5 (available on: http://www.megasoftware.net) (1000 bootstrap replicates).

Figure 3

Transcription activation of CmMYB19 in yeast. (a) the orientation of the plate. (b) the growth of transformed yeast cells. Cells harboring pCL1 were able to grow on the SD/-His-Ade medium (SD, Synthetic Dropout Media), while those containing pGBKT7 could not.

Figure 4

Sub-cellular localization of CmMYB19 expression in transiently transformed onion epidermal cells. Bar: 100 μm.

Figure 5

Transcriptional changes of CmMYB19 in various organs of the chrysanthemum plant, as assayed by qRT-PCR.

Figure 6

Transcriptional changes of CmMYB19 following aphid infestation or mock puncture treatment, as assayed by qRT-PCR. At each time point, different letters (a, b, c) designate significantly different expression levels between control, aphid infestation and puncture (p < 0.05).

Figure 7

Transgenic chrysanthemum lines over-expressing CmMYB19 inhibit the multiplication of M. sanbourni aphids. (a) Amplification of fragment of Hyg^r gene. M, molecular marker, non-transgenic, CmMYB19-ox-1, CmMYB19-ox-2 were non-transgenic plants and CmMYB19 overexpressing plants, respectively; (b) CmMYB19 transcript abundance in CmMYB19 over-expressing and non-transgenic plants; (c) the number of aphids present on CmMYB19 over-expressing and non-transgenic plants measured at 3–21 d (days) after infestation; and (d) differential proliferation of aphids between CmMYB19 over-expressing and non-transgenic plants.

Figure 8

Lignin content determined in CmMYB19 over-expressing and non-transgenic plants. a, b, c represent significantly different expression levels between non-transgenic and CmMYB19 overexpressing plants (p < 0.05).

Figure 9

The transcription of genes involved in lignin synthesis in CmMYB19 over-expressing and non-transgenic plants, as assayed by qRT-PCR. For each gene, different letters (a, b, c) represent significantly different expression levels between non-transgenic and CmMYB19 overexpressing plants (p < 0.05).