CONFERENZA-OBIETTIVO 2030 - 12-13/12/2019

Gene Silencing in plant parasitic nematodes and their long-term effects

Francesca De Luca, Elena Fanelli, AlbertoTroccoli,

Types of targets for control

Pratylenchus vulnus endoglucanasesGenes coding for endoglucanases belong to the family of cell walldegrading enzymes and ten genes are present in P. vulnus. Five of themwere molecularly and functionally characterized in our lab:

Pv-eng-1

Pv-eng-2

Pv-eng-3

Pv-eng-5

Pv-eng-8

in situ hybridization

Pv-eng-1

Pv-eng-2

qRT-PCR

Pv-eng-5

Expression and localization

XII Convegno Nazionale SIN - 26-28/09/2016

Expression and localization

Pv-eng-8

qRT-PCR

in situ hybridization

T7

T7

•Due separate reazioni di amplificazione

•Due separate reazioni di trascrizione

RNAAnnealing

dsRNA

Soaking nematode in ds RNA

Analisi funzionale di Pv-eng-1, Pv-eng-5 e Pv-eng-8mediante RNAi

Phenotypic effects after soaking of Pratylenchus vulnus

Substantial reduction in vitro in target gene expression(pv-eng-1 more susceptible to RNAi than pv-eng-8 and pv-eng-5

98%

57%

62%

10 treated males and 20 treated females with dsRNA were inoculated on carrot discs Incubation at 23°C for 33 days.

Nematodes Count atstereo microscopy to determine the effectsof soaking

Life cycle and reproduction

Silencing of Pv-eng-1 and Pv-eng-8 significantlyreduce reproduction and determine a slowdown of lifecycle.

Pratylenchus loosi

Caratterizzazione del gene per la β-1,4-endoglucanasi Pl-eng-2 nel nematodePratylenchus loosi, uno dei più pericolosifitoparassiti del tè diffuso in tutto ilmondo, soprattutto in Giappone, SriLanka, India, Cina, Bangladesh ed Iran

Expression and localization of Pl-eng-2

qRT-PCR

in situ hybridization

Silencing of Pl-eng-2 significantly reduces reproduction

Conclusions

RNAi is a powerful technology to study gene function in plantparasitic nematodes and to silence a specific gene;

Silencing of Pv-eng-1, Pv-eng-5 and Pv-eng-8 resulted in reduction oftarget gene expression at different level; dsRNA Pv-eng-1 and dsRNAPv-eng-8 treated nematodes showed a slowdown of life cycle andreduction of reproduction;

Cell-wall degrading enzymes could be attractive targets forengineering crop plants for nematode control;

Host Induced Gene Silencing via RNAi seems to be the mostpromising strategy for the control of plant parasitic nematodes infuture.

Diapositiva numero 1Diapositiva numero 2Diapositiva numero 3Diapositiva numero 4Diapositiva numero 5Diapositiva numero 6Types of targets for controlPratylenchus vulnus endoglucanasesDiapositiva numero 9Diapositiva numero 10Diapositiva numero 11Diapositiva numero 12Diapositiva numero 13Diapositiva numero 14Diapositiva numero 15Diapositiva numero 16Pratylenchus loosiDiapositiva numero 18Diapositiva numero 19Diapositiva numero 20