Biotechnology approaches towards the control of the root lesion nematode Pratylenchus penetrans (PRATYTECH)

Worldwide crop losses due to plant-parasitic nematodes have been estimated at $118 billion annually, with root lesion nematodes

(RLN), Pratylenchus spp., ranking third in terms of economic losses. The economic impact of Pratylenchus spp. is due to their wide

host range, comprising more than 400 plant species, including agronomically important crops, ornamentals/floral plants, and many

fruit and nut trees. Surveys conducted in Portugal revealed the presence of different species of Pratylenchus associated with

important crops. In recent studies, several RLN species were isolated and identified from potato root samples collected in the main

potato production regions. Pratylenchus penetrans was the most abundant species, followed by P. neglectus, P. crenatus and P.

thornei, which were detected for the first time associated with potato in Portugal. More recently, a new species of Pratylenchus has

been found associated with severe damage caused on the production of bulb flowers (e.g. Amaryllis) in several greenhouses in

Portugal, reinforcing the dimension of the threat these species can impose to agriculture and floriculture systems. Host resistance to

Pratylenchus spp. is very limited, as only a few loci have been linked to resistance/tolerance to some RLN species. Effective and

long-lasting control strategies based on currently chemical compounds are hampered by increasing regulations because most

nematicides are non-specific, notoriously toxic and pose a potential threat to the soil ecosystem, ground water and human health. A

research area that is currently very promising is the identification of critical metabolic and parasitism-related genes of the plant

pathogen, which silencing through RNA interference (RNAi) promotes lethal or inhibitory effects on its own development. However,

one imperative action is the identification of the plant pathogen specific genes lacking homologs in non-target organisms. Promising

results controlling several plant-parasitic pathogens through RNAi-mediated gene silencing, includes those from bacteria, fungi,

insects, oomycetes and nematodes, mainly for root-knot (Meloidogyne spp.) and cyst nematodes (Globodera and Heterodera spp.).

Although the successful silencing of genes using RNAi technology has been demonstrated for some Pratylenchus species, only a few

genes have been tested (e.g. pat-10, unc-87, Pv010 and eng-1), indicating oversight for this important group of plant parasites.

The main goal of this project is to identify protein-coding genes in P. penetrans that could be established as new nematode targets

for the development of more specific and efficient crop management strategies. Global parasite-host transcriptomes constitute an

excellent tool to provide a general overview of the molecular dialogue established between the pathogen and the host, and

ultimately for the identification of the main host molecular pathways and individual genes involved in such interaction. Our starting

material for this project consist of different in house sets of large-scale transcriptome data (paired-end sequencing mRNA-seq,

Illumina) generated for P. penetrans: 1) from the nematode alone, and 2) from infecting soybean plants with this species, which will

be use for the selection of new nematode gene targets. In addition, new sets of transcriptome data will be obtained for P. penetrans

infecting potato. In this context, this project engages the implementation of biotechnological approaches for crop improvement;

using mRNA-seq Illumina data for the identification of new nematode target genes and RNAi-mediated nematode gene silencing for

studying their anti-nematode activity. The strategy used during this project includes the study of the levels of expression and tissue

localization of the nematode gene to be silenced, and consequently generation of siRNA and dsRNA constructs for RNAi-mediated

gene silencing, via nematode soaking assays, and posteriorly direct delivery via in planta, to evaluate its efficiency on the nematode

development disruption or lethality. Another relevant aspect of the PratyTech proposal is the focus on the elucidation of the host

gene expression profile, and cellular changes, upon P. penetrans infection in potato, providing important insights into the molecular

mechanisms involved in such economic important crop. In the long-term, our goal is to produce alternative and innovative

methodologies aiming crop resistance against RLN applied to important floral/agriculture/orchard crops, which will be pertinent not

only for Portugal, but also in other areas where P. penetrans have an economic impact.

3.2. Descrição Técnica

3.2 Technical