Virus-Induced Gene Silencing in Chrysanthemum seticuspe Using the Tomato Aspermy Virus Vector

Abstract

Chrysanthemum is one of the most economically important flowers globally due to its high ornamental value. In recent years, a large percentage of the Chrysanthemum seticuspe genome has been determined, making this species useful as a model chrysanthemum plant. To fully utilize the genome's information, efficient and rapid gene functional analysis methods are needed. In this study, we optimized the tomato aspermy virus (TAV) vector for virus-induced gene silencing (VIGS) in C. seticuspe. Conventional plant virus inoculation methods, such as the mechanical inoculation of viral RNA transcripts and agroinoculation into leaves, did not achieve successful TAV infections in C. seticuspe, but vacuum infiltration into sprouts was successful without symptoms. The TAV vector harboring 100 nucleotides of the phytoene desaturase (PDS) gene caused photobleaching phenotypes and a reduction in CsPDS expression in C. seticuspe. To our knowledge, this is the first report of VIGS in chrysanthemums.

Keywords: chrysanthemum; tomato aspermy virus; virus-induced gene silencing.

Figure 1

Efficient inoculation method of tomato aspermy virus (TAV) into Chrysanthemum seticuspe. (a) RT–PCR detection of TAV in C. seticuspe inoculated with the TAV cDNA clone. M, DNA size marker; the transcripts, leaf infiltration, and SVI indicate that RNA was extracted from the C. seticuspe inoculated by the mechanical inoculation of viral transcripts, agroinoculation into leaves, and vacuum infiltration into sprouts, respectively; healthy, healthy plant; positive control, RNA extracted from the Nicotiana benthamiana inoculated with TAV-ChJ. (b) C. seticuspe plants that the TAV was detected at 28 days postinoculation (dpi) (SVI). Mock plants were inoculated with a pJL89 binary vector by SVI. Bars, 10 mm.

Figure 2

Virus-induced gene silencing (VIGS) induction in Chrysanthemum seticuspe by the tomato aspermy virus (TAV) vector. (a) Schematic representation of the VIGS constructs. The partial 100 nucleotides of the C. seticuspe phytoene desaturase (PDS) gene were introduced just downstream of the capsid protein (CP) open reading frame in TAV RNA3 (pJL89T3_CsPDS100) and agroinoculated with pJL89T1 and pJL89T2. The TAV genomes were cloned between the 35S promoter (35S) and the nopaline synthase terminator (NOS). The ribozyme sequence of hepatitis delta virus ribozyme (Rz) is located just downstream of the 3′ untranslated region of TAV cDNA. (b) C. seticuspe infected with the TAV vector described in Panel A at 28 days postinoculation (dpi). The photobleaching areas are indicated by white arrowheads. (c) RT–PCR analysis of the TAV vector harboring the partial CsPDS sequence. RNA samples were extracted from C. seticuspe leaves at 28 dpi. pJL89 was used as a negative control. TAV not harboring the partial CsPDS sequence was used as the empty vector control. The RT–PCR product of the TAV vector harboring the partial CsPDS sequence was approximately 1200 base pairs (bp), whereas that of the TAV empty vector was approximately 1100 bp. (d) RT–qPCR analysis of CsPDS expression levels. RNA samples were extracted from C. seticuspe leaves at 28 dpi. TAV not harboring the partial CsPDS sequence was used as the empty vector control. Each RT–qPCR analysis was performed with five to six biological replicates. Boxes show the interquartile ranges including 25–75% of the values, and whiskers indicate the highest and lowest values of data. Horizontal lines and cross marks in boxes indicate the medians and means, respectively. The value outside 1.5 times the interquartile range between 25% and 75% of each group was considered an outlier and indicated with a dot. Asterisks indicate significant differences by Student’s t-test at p < 0.05.