A motif to unveil mechanisms of parasitism gene regulation in the pinewood nematode as a target for disease control and plant resistance.

Plant pathogens cause severe economic and ecological problems in a wide range of crops and forestry plant species worldwide. Crop losses due to pests and diseases are a major obstacle towards achieving global food security. Annual crop losses attributed to nematodes are estimated in billions of dollars a year(1). In forests, pathogens can affect sustainable management by affecting economic trade and serious ecological losses can occur(2). Surveys conducted in Portugal showed that the endoparasitic and EU quarantine pinewood nematode (PWN), Bursaphelenchus xylophilus is the major cause for the tremendous decline of the maritime pine (Pinus pinaster)-Pine wilt disease(3). The host range of susceptible species to PWN also includes P.nigra, P.sylvestris. As damage caused by this nematode in pine trees (several Pinus spp.) is more severe at high temperatures, it has been suggested that climate change is likely to increase the problems caused by PWN worldwide(4-6). Restricted use of pesticides, and other chemicals for agriculture due to EU directives requires new, integrative and sustainable control solutions(7). However, the challenge persists: to find new solutions in sustainable agriculture and forestry systems, it is crucial to understand the mechanisms by which these organisms parasite plants. The interactions of plant-parasitic nematodes (PPN) with their plant hosts are mediated by parasitism proteins (effectors): secreted proteins produced by the pathogen that delivered into the host and interact with plant machinery to promote disease. PPNs use hundreds of effectors to manipulate their host. The improvement of plant resistance to PPN can be achieved by identifying effectors and functionally modify or block them towards an effect in the nematode success infecting the host(8). NemaWAARS proposal focusses on a broader question: how is parasitism regulated and which mechanisms control the gene expression of effectors? And by understanding the molecular interaction will it allow us to disrupt it, helping plant immunity? Following a recent out breaking discovery associated to the PWN parasitism genes, supported by genomic (9) and transcriptomic data (10,11) previously established for this pathogen and a DNA motif, STATAWAARS, associated in the promotor region of secreted effector genes of PWN was identified(12). Given that this non-coding genetic signature unifies many sequences of unrelated effectors, it implies the existence of a potential major regulator(s), that binds to this sequence to control the expression of downstream effector genes. A similar master regulator has been proposed for other PPN not related to migratory nematodes(14-17). We hypothesize that disrupting this regulator(s), it would disrupt simultaneously the expression of a large number of associated effector genes. This a new attractive focus for host induced gene silencing, as switching-off the regulator of several effectors at the same time, can potential reduce and inhibit the performance of this pathogen. Building on genomic and transcriptomic information that we have developed for this pathogen and using an approach pioneered in other plant-parasitic nematodes, this proposal aims to (i) identify transcription regulators that are associated with these motifs (in vivo) and enriched in the gland cell tissues; (ii) understand how candidate transcription factors have an effect in PWN parasitism (functional validation). The discovery of STATAWAARS motif opened the door to a first comprehensive understanding of how a parasitism is regulated in these nematodes. The strategy in NemaWAARS will include an innovative approach of a modified CRISPR-Cas9 system (21) to explore the molecules that govern effector gene expression in vivo, applied in PWN research. More, the study of the levels of expression and tissue localization of the nematode genes to be silenced, generation of dsRNA constructs for RNAi-mediated gene silencing, transcriptomic and proteomics analysis and evaluation of its efficiency on the nematode development in the plant host(in vitro;26) will also be addressed. Is expected to obtain new insights in the proteins and RNAs that are associated to effector promoters, as a regulatory network that, once disrupted, will be a totally unexplored area in PWN group. Other PPN that have a similar infection strategy and cause huge damage to important economic crops will benefit from these outputs and are currently being explored under an ongoing international collaborative network. The project joins a solid and multidisciplinary national research team. NemaWAARS will provide new control targets and strategies that could be attractive and potentially used in industry and explore sustainable solutions for improved forestry management in line with the 2nd and 15th United Nations Goals and European Green Deal goals. Therefore, NemaWAARS will contribute to promote agronomic and forestry ecosystem sustainability and avoid biodiversity loss.