Optimizing virus-induced gene silencing efficiency with Cymbidium mosaic virus in Phalaenopsis flower

Ming-Hsien Hsieha,b, Hsiang-Chia Luc, Zhao-Jun Pana, Hsin-Hung Yehc, Shyh-Shyan Wangb, Wen-Huei Chend, Hong-Hwa Chena,d,
Plant Science, November 2012

Introduction
Orchidaceae is one of the largest families of angiosperms and includes approximately 25,000 species in 900 genera. The genus Phalaenopsis is a popular pot plant because of its graceful appearance, long-lasting flowering and wide variety of species. The molecular genetic information of orchids is still lagging behind Arabidopsis and rice. Therefore, functional genomics tools are needed to accelerate the discovery of genes involved in floral traits of Phalaenopsis

 The Cymbidium mosaic virus-based VIGS vector (pCymMVgateaway) has been used to induce gene silencing in orchids.
Targets gene - PeUFGT3 : encodes an enzyme involved in anthocynin biosynthesis for red orchid flowers - PeMADS5 : B-class MDS- box gene, involved in orchid floral morphogenesis - PeMADS6 : B-class MDS- box gene ,involved in orchid floral morphogenesis Optimization -length of the inserted DNA fragment -inoculation sites -effect of development maturation status of inflorescence.

Methods
Plants materials Construction of pCymMVGateway plasmids Agro-inflitration of plants

Statistical analysis

Determination of anthocyanin content

Real-time quantitative RTPCR

Results
1.0 Effect of DNA length in silencing efficiency of PeUFGT3 gene Figure 1. A Position and length of insert fragment for silencing

- Various length of PeUFGT3 cDNA ( 1498, 1113, 758, 334, 81 bp) - Plasmid designated as PeUFGT3 -1 to PeUFGT3-5
Figure 1.B Inflorescence of Phalaenopsis Figure 1.C mock inoculated flower compared with PeUFGT3 silenced plant (1-5) - Discolorations appeared in sepals and petals of PeUFGT3 3- 5 - Discoloration was significant when the shortest fragment (81bp) was used, lip showed little discoloration.

- the rate of anthocyanin content between mock-treated and PeUFGT3-1-to PeUFGT3-5-silenced flowers was 59.3%, 58.1%, 53.5%, 38.7% and 31.5%, with the mocktreated flowers as 100%
- Shortest fragment showed lowest anthocyanin content - mRNA level of PeUFGT3 was reduced to 23.6%, 22.3%, 16.7%, 9.3% and 4.2% for plants with silenced PeUFGT3-1 to PeUFGT3-5, respectively.

the shortest insert fragment produced the highest genesilencing efficiency, with no significant differences in silencing effect or mRNA level between 81and 334-bp fragments

2.0 Effect of fragment location of PeUFGT3 gene

- cloned three non-overlapping fragments of PeUFGT3 into pCymMV-Gateway.

- the flowers of PeUFGT3-6, PeUFGT3-7-, PeUFGT3-8 and PeUFGT3-4 silenced plants showed discolored sepals and petals, and had a white spot on the abaxial edge of lips.

- All constructs produced similar reductions in both anthocyanin content and relative PeUFGT3 mRNA abundance. - no differential silencing efficiency among various PeUFGT3 DNA fragments selected for VIGS.

3.0 Effect of cDNA insert length and location on silencing of PeMADS5 gene

- Three regions, including the MADSbox region, the carboxyl terminal region and the 3-untranslated region.

- Only the PeMADS5-2 fragment caused phenotype changes, including dark green coloration on the abaxial surface of the sepals. - discoloration on abaxial surface of the lateral sepals ( Figure 3C) - Reduced floral organ size (Figure 3E) - The dark green coloration appeared on the edges of the abaxial side of the sepals and usually on the first to fourth flower in the inflorescence.

- The mRNA levels in sepals of PeMADS5-1- to PeMADS5-4-silenced flowers were reduced to 24.1%, 0.4%, 13.4% and 11.6%, respectively, in sepals. - The PeMADS5 mRNA levels in the petal and lip were also reduced but not as profoundly as in the sepal.

4.0 Effect of cDNA insert length and location on silencing of PeMADS6 gene - constructed various insert fragments of PeMADS6 cDNA fragments, including 4702 nt (699 bp), 488 nt (85 bp), 417497 nt (81 bp), and 622702 nt (81 bp), designated PeMADS6-1 to PeMADS6-4, respectively

- Flowers of PeMADS6-1-and PeMADS6-2-silenced plants were unable to fully expand. (Fig. 4B)
- showed discolored areas in the lip (Fig. 4C) - PeMADS6-1and PeMADS6-2silenced flowers, showed dark green stripes or areas. May Have been affected by non trageting silencing of other B-class gene.

- PeMADS6-1- to PeMADS6-4-silenced flowers showed reduced mRNA levels of PeMADS6 to 7.4%, 3.0%, 26.2% and 22.9%, respectively, in sepals; 18.1%, 8.8%, 35.5% and 35.3%, respectively, in the petal; and 8.7%, 1.3%, 15.1% and 12.6%, respectively, in the lip. - PeMADS6- 1 and PeMADS6-2 both showed lowest mRNA levels.

4.0 VIGS phenotypes with Agro-inflitration of type I and type II inflorescences

- tested two different developmental maturation states of inflorescence. - Type I inflorescences contained six visible internodes with no visible floral buds. - type II inflorescences were more mature, containing eight visible internodes with one visible floral bud - silencing PeMADS6-2 in the type II inflorescence, the first three floral buds were unable to bloom normally and remained closed.

- The distal ends of the sepals were bonded together and did not detach , the size of floral organs was reduced and the morphogenesis of tepals was significantly affected in both inflorescences.

- The mRNA level was reduced to 19.9%, in type II inflorescence which is much lower than the type I inflorescence (40.6%)

5.0 Comparison of Agro-inflitration injection position leaf or inflorescence

- Vector inoculated by injecting the emerging inflorescence and also injecting the leaf right above the emerging inflorescence.

- The VIGS efficiency and VIGS phenotypes were similar in both treatments. - Injection time spent per plant for leaf and inflorescence injection was 1 min and 25 min, respectively. - Agro-inflitration in leaves is a time saver and produce less damage to flower bud.

Discussion
1. The DNA fragment size used for silencing can be as small as 7885 bp in Phalaenopsis. - Vectors with large inserts are more susceptible to losing the foreign insert through recombination in the plant compared to vectors with small inserts. - To reduce the burden of the viral vector in VIGS delivery, minimizing the silencer size is important. 2. Effect of location of the cDNA fragment on silencing efficiency varies among genes. - Different location of inserted coding region of B-class MADS-box genes (PeMADS5 and PeMADS6) showed VIGS phenotypes changed but not in PeUFGT3. 3. Knocking down PeMADS6 cDNA caused an uncharacterized phenotype. 4. VIGS phenotype was caused by silencing target genes, paralogous genes and nontarget genes

5. Developmental maturation status of inflorescence affected silencing efficiency

- The PeMADS6-2 silencing efficiency was greater in more mature type II inflorescences of Phalaenopsis plants containing eight internodes and one visible floral bud than in younger type I inflorescences with six internodes and without visible floral buds. 6. Similar silencing efficiency obtained for different distances of Agro-infiltration sites - mRNA levels in PeMADS6- silenced plants did not differ with leaf- and inflorescence injection inoculation. - For Agroinfiltration, leaf injection saves time and causes less damage to inflorescences.

Conclusion
B-class MADS-box gene silencing with the 5 terminus of the coding region produced a VIGS phenotype in PeMADS5 but a more severe phenotype in PeMADS6. However, the silencing of these genes could co-suppress several members of the gene family and overcome a possible functional redundancy when using regions that are conserved between genes. Agro-infiltration in an inflorescence containing eight internodes with one visible floral bud could improve the VIGS method in floral functional genomics studies of orchids. With Agro-infiltration, leaf rather than inflorescence injection saves time and causes less damage to inflorescences.