Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Jul 16;8(7):e69463.
doi: 10.1371/journal.pone.0069463. Print 2013.

Molecular characteristics and efficacy of 16D10 siRNAs in inhibiting root-knot nematode infection in transgenic grape hairy roots

Yingzhen Yang  1 , Yingyos Jittayasothorn, Demosthenis Chronis, Xiaohong Wang, Peter Cousins, Gan-Yuan Zhong
Affiliations

Molecular characteristics and efficacy of 16D10 siRNAs in inhibiting root-knot nematode infection in transgenic grape hairy roots

Yingzhen Yang et al. PLoS One. .

Abstract

Root-knot nematodes (RKNs) infect many annual and perennial crops and are the most devastating soil-born pests in vineyards. To develop a biotech-based solution for controlling RKNs in grapes, we evaluated the efficacy of plant-derived RNA interference (RNAi) silencing of a conserved RKN effector gene, 16D10, for nematode resistance in transgenic grape hairy roots. Two hairpin-based silencing constructs, containing a stem sequence of 42 bp (pART27-42) or 271 bp (pART27-271) of the 16D10 gene, were transformed into grape hairy roots and compared for their small interfering RNA (siRNA) production and efficacy on suppression of nematode infection. Transgenic hairy root lines carrying either of the two RNAi constructs showed less susceptibility to nematode infection compared with control. Small RNA libraries from four pART27-42 and two pART27-271 hairy root lines were sequenced using an Illumina sequencing technology. The pART27-42 lines produced hundred times more 16D10-specific siRNAs than the pART27-271 lines. On average the 16D10 siRNA population had higher GC content than the 16D10 stem sequences in the RNAi constructs, supporting previous observation that plant dicer-like enzymes prefer GC-rich sequences as substrates for siRNA production. The stems of the 16D10 RNAi constructs were not equally processed into siRNAs. Several hot spots for siRNA production were found in similar positions of the hairpin stems in pART27-42 and pART27-271. Interestingly, stem sequences at the loop terminus produced more siRNAs than those at the stem base. Furthermore, the relative abundance of guide and passenger single-stranded RNAs from putative siRNA duplexes was largely correlated with their 5' end thermodynamic strength. This study demonstrated the feasibility of using a plant-derived RNAi approach for generation of novel nematode resistance in grapes and revealed several interesting molecular characteristics of transgene siRNAs important for optimizing plant RNAi constructs.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: PC contributed to this work while he was an employee with the United States Department of Agriculture-Agricultural Research Service Grape Genetics Research Unit in Geneva, New York. The work described in the manuscript has no connection with PC’S current work or employer (E & J Gallo Winery). This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Representative transgenic grape hairy root lines used in this study.
Individual hairy root lines carrying a 16D10 RNAi construct, pART27-42 or pART27-271, were cultured and inoculated with J2 RKNs to evaluate their resistance against RKNs. pART27 0 was a control line which was transformed with an empty binary vector pART27. Note that pART27-271 line 20 and pART27-42 line 24 showed contrasting variation in their root morphology and proliferation. The pictures were taken three weeks (pART27-271 line 20 and pART27-42 line 24) or five weeks (the rest) after nematode inoculation.
Figure 2
Figure 2. Reproduction of root-knot nematodes on 16D10 transgenic hairy root lines.
(A) Eggs per hairy root. (B) Eggs per gram hairy root. NC is the negative control (pART27 0). 42-L1 and 271-L5 represent the abbreviations of pART27-42 line 1 and pART27-271 line 5, respectively. Bars represent the means±SEs observed from individual hairy root lines. Bars (hairy root lines) with “Δ” were significantly different from the negative control at P<0.01, on the basis of log10 transformed data. Data for this figure were provided in Table S1.
Figure 3
Figure 3. Distribution of the 16D10 small RNAs along the pART27-42 hairpin stem.
The hairpin structure of the pART27-42 construct (the spliced-out intron not included) is presented with the 16D10 passenger ssRNAs aligned along the sense strand (above) and the guide ssRNAs aligned along the antisense strand (below). The relative small RNA abundance is graphically represented by the relative thickness of a block/line. Due to the limitation of graphic resolution, only those small RNAs with more than 200 reads were presented. The blocks with color variation in the 3′ ends indicate presence of mismatches. The “.” at the stem base represents the 5′ and 3′ overhangs due to the presence of cloning sites and other residual sequences from the pART27-42 construct. The green arrows pointed to the first putative dicer cleavage site (21 nts away from the 5′ residue) and the red arrows pointed to the second putative dicer cleavage site (21 nts away from the first putative cleavage site). The grey boxes highlighted the siRNA duplex produced by these two cleavage events. Data for this figure were provided in Table 2 and Table S2.
Figure 4
Figure 4. Distribution of the 16D10 small RNAs along the pART27-271 hairpin stem.
The schematic hairpin structure of the pART27-271 construct is presented with the green line representing the sense strand, the red line representing the antisense strand, and the blue open circle representing the 39 nt loop. The numbers “100” and “200” along the stem indicate nucleotide positions from the 5′ stem end. The 16D10 42 bp core coding region is marked as a purple box on the stem. GC content was marked for the stem base region, the middle core region, and loop terminus region. Each small RNA is represented as a block/line, with the thickness of a block/line indicating the relative abundance of a particular small RNA. Passenger and guide ssRNAs were aligned along the sense and antisense strands, respectively. Data for this figure were provided in Table S4.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Nicol JM, Stirling GR, Rose BJ, May P, Van Heeswijck R (1999) Impact of nematodes on grapevine growth and productivity: current knowledge and future directions, with special reference to Australian viticulture. Australian Journal of Grape and Wine Research 5: 109–127.
    1. Cain DW, Mckenry MV, Tarailo RE (1984) A new pathotype of root-knot nematode on grape rootstocks. J Nematol 16: 207–208. - PMC - PubMed
    1. Cousins P, Johnston D, Switras-Meyer S, Boyden L, Vidmar J, et al. (2007) USDA ARS research in grape rootstock breeding and genetics. Acta Hort (ISHS) 733: 51–58.
    1. Cousins P, Walker MA (2002) Genetics of resistance to Meloidogyne incognita in crosses of grape rootstocks. Theor Appl Genet 105: 802–807. - PubMed
    1. Xu K, Riaz S, Roncoroni NC, Jin Y, Hu R, et al. (2008) Genetic and QTL analysis of resistance to Xiphinema index in a grapevine cross. Theor Appl Genet 116: 305–311. - PubMed

Publication types

Substances

Grants and funding

This project was supported by the United States Department of Agriculture-Agricultural Research Service Project 1910-21220-004-00D. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.