Optimization and Validation of PpPDS Gene Silencing Induced by Prunus Necrotic Ring Spot Virus in Prunus persica

doi:10.16420/j.issn.0513-353x.2022-0484

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (7): 1587-1600.doi: 10.16420/j.issn.0513-353x.2022-0484

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Optimization and Validation of PpPDS Gene Silencing Induced by Prunus Necrotic Ring Spot Virus in Prunus persica

PAN Jiajia, ZHANG Dongmei, MENG Jian, GAO Sunan, ZHU Kaijie, LIU Junwei^*(), LI Guohuai

National Key Lab for Germplasm Innovation and Utilization of Horticultural Crops，College of Horticulture and Forestry Sciences，Huazhong Agricultural University，Wuhan 430070，China

Received:2022-12-30 Revised:2023-04-06 Online:2023-07-25 Published:2023-07-26
Contact: LIU Junwei

Abstract

Abstract:

At present，VIGS still works as a main method for gene silencing studies in peach. However，the current VIGS efficiency is low that is urgent to develop a more efficient VIGS system in peach. Using PpPDS（Phytoene desaturase gene）as the indicator gene，the effects of infection mode，viral vector，temperature，age of inoculated seedling and bacterial concentration on the VIGS efficiency in peach leaves，were comprehensively investigated. The results showed that leaf agroinfiltration was an effective way of infection；Prunus necrosis ring spot virus（PNRSV）vectors（pCaRNA1/2 and pCaRNA3）were better than the modified TRV vectors（pTRV1 and pTRV2）of tobacco rattle virus；the infection temperature was better at 22 ℃；peach seedlings with 5-6 fully expanded leaves were higher，reaching 85% of gene silencing efficiency；the bacterial concentration at OD₆₀₀= 1.0 was better. Based on the optimized parameters of VIGS system，two peach chlorophyll biosynthetic genes（PpGluTR and PpPOR）were selected as target to verify the optimized VIGS system. The results showed that the expressions of PpGluTR and PpPOR were significantly repressed，and the change of leaf color was evident. The studies suggest that the optimized VIGS system has good stability and high silencing efficiency，which can be used for gene silencing studies in peach.

Key words: Prunus persica, virus-induced gene silencing（VIGS）, Prunus necrotic ring spot virus （PNRSV）, phytoene desaturase, chlorophyll biosynthetic gene

PAN Jiajia, ZHANG Dongmei, MENG Jian, GAO Sunan, ZHU Kaijie, LIU Junwei, LI Guohuai. Optimization and Validation of PpPDS Gene Silencing Induced by Prunus Necrotic Ring Spot Virus in Prunus persica[J]. Acta Horticulturae Sinica, 2023, 50(7): 1587-1600.

Figures/Tables 13

Table 1 Primer sequences used in this study

引物	目的	序列（5′-3′）	条带大小/bp
Primer	Purpose	Sequence	Product size
1	PpPDS CDS	F：ATGTCTCAGTGGGCTTGTGTCTCTG；R：TCACCGAACGCTTGCCTCAGCCACC	1 722
2	PpPDS	F：GCTCTAGAAGAAAGCTGAAGAACACAT；R：GCTCTAGAGATTGTAATATTCCTTACAT	100
3	pCaRNA3-PpPDS	F：GCTTCCCTAACGGGGCATCC；R：AGGTCTTGGTTAGGGATTTG	375
4	PpGluTR	F：AAGACCTGCTTGAGATCTAGAGAGAATGTTGGTGATTGGTGCTG； R：GTGTGCTTATCTCACTCTAGATGTTGATATTTCTGTCCCTAGGAATG	320
5	PpPOR	F：AAGACCTGCTTGAGATCTAGATTGATGTGCTTGTGTGCAATGC； R：GTGTGCTTATCTCACTCTAGACAAGGTGTTTGTGTTCCCTGTAAT	209
6	pCaRNA3	F：GCTTCCCTAACGGGGCATCC；R：AGGTCTTGGTTAGGGATTTG	269
7	pTRV1	F：TTACAGGTTATTTGGGCTAG；R：CCGGGTTCAATTCCTTATC	647
8	pTRV2	F：TACGACGAACCAAGGG；R：TGCGAAACTCAAATGCT	427
9	PpPDS qRT-PCR	F：CCTTGCAAAGATCTCCCCTA；R：CGAGCAACAAGCAATTCGTA	145
10	PpGluTR qRT-PCR	F：GGAAGATGGGGAAGCTTGTGA；R：GGTTTCTGATGCCGTGCTTG	200
11	PpPOR qRT-PCR	F：TTGTGTGCAATGCTGCTGTT；R：CGAAAGAAGGAAGTGCCCGA	110
12	PpTEF2 qRT-PCR	F：AGCAAGTCACCCAACAAGCATA；R：CCAACCAAACTCTTCAGCCAAT	147

Fig. 1 Schematic illustration of peach seedlings sampling ①Phenotypic upper leaf：The leaves above the phenotypic leaves without phenotypic appearance；②Phenotypic leaf：The leaves with phenotypic appearance；③Phenotypic lower leaf：The leaves below the phenotypic leaves without phenotypic appearance；④Injected leaf：The agroinfiltrated leaves；⑤Stem：the above-ground part left after cutting off the leaves；⑥Root：underground parts after washing culture soil.

Fig. 2 PCR amplification of peach PpPDS gene（A）and positive test of recombinant plasmid pCaRNA3-PpPDS（B）

Fig. 3 PCR analysis of pCaRNA3 virus and pTRV virus infection into peach seedlings M：Marker；1：Blank control（non-infected）；2：Negative control（empty vector）；3：PDS silencing.

Fig. 4 Silencing phenotypes of peach seedlings agroinfiltrated with pCaRNA3-PpPDS and pTRV2-PpPDS

Fig. 5 Effect of different temperature（A）and seedlings age（B）on VIGS efficiency in peach ‘nd’ stands for not detected；Different lowercase letters on top of bars indicate statistical significance at P < 0.05 among different treatments.

Fig. 6 Silencing effect of different peach leaves agroinfiltrated with pCaRNA3-PpPDS and pTRV2-PpPDS The upper leaves of phenotypic leaves of pCaRNA3-PpPDS were all phenotypic leaves, so here no phenotypic upper leaf.

Fig. 7 Transcriptional abundance of PpPDS in different parts of peach seedlings agroinfiltrated with pCaRNA3-PpPDS（A），pTRV2-PpPDS（B）and their empty vectors Asterisks indicate statistical significance between treatment and negative control（* α = 0.05 and ** α = 0.01）.

Fig. 8 Silencing efficiency of pCaRNA3-PpPDS at different bacterial concentrations in peach seedlings Different lowercase letters on top of bars indicate statistical significance at P < 0.05 among different treatments. The same below.

Fig. 9 Leaf morphology of peach seedlings agroinfiltrated with pCaRNA3-PpGluTR and pCaRNA3-PpPOR after 28 days

Fig. 10 Chlorophyll fluorescence of peach leaves agroinfiltrated with pCaRNA3-PpGluTR and pCaRNA3-PpPOR Symbol-1，-2 and -3 indicate three biological replicates.

Fig. 11 Chlorophyll content of peach leaves agroinfiltrated with pCaRNA3-PpGluTR and pCaRNA3-PpPOR

Fig. 12 Transcript levels of PpGluTR and PpPOR in PNRSV-mediated VIGS system in peach seedlings

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Optimization and Validation of PpPDS Gene Silencing Induced by Prunus Necrotic Ring Spot Virus in Prunus persica

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