Tomato Response to Fusarium spp. Infection under Field Conditions: Study of Potential Genes Involved

Tomato is one of the most important horticultural crops in the world and is severely affected by Fusarium diseases. To successfully manage these diseases, new insights on the expression of plant– pathogen interaction genes involved in immunity responses to Fusarium spp. infection are required. The aim of this study was to assess the level of infection of Fusarium spp. in field tomato samples and to evaluate the differential expression of target genes involved in plant–pathogen interactions in groups presenting different infection levels. Our study was able to detect Fusarium spp. in 16 from a total of 20 samples, proving the effectiveness of the primer set designed in the ITS region for its detection, and allowed the identification of two main different species complexes: Fusarium oxysporum and Fusarium incarnatum-equiseti. Results demonstrated that the level of infection positively influenced the expression of the transcription factor WRKY41 and the CBEF (calcium-binding EF hand family protein) genes, involved in plant innate resistance to pathogens. To the best of our knowledge, this is the first time that the expression of tomato defense-related gene expression is studied in response to Fusarium infection under natural field conditions. We highlight the importance of these studies for the identification of candidate genes to incorporate new sources of resistance in tomato and achieve sustainable plant disease management.This research was supported by projects “Development of a new virus-based vector to control TSWV in tomato plants” with the references ALT20-03-0145-FEDER-028266 and PTDC/ASP-PLA/28266/2017, and “Control of olive anthracnose through gene silencing and gene ex- pression using a plant virus vector” with the references ALT20-03-0145-FEDER-028263 and PTDC/ASP- PLA/28263/2017, both projects co-financed by the European Union through the European Regional Development Fund, under the ALENTEJO 2020 (Regional Operational Program of the Alentejo), ALGARVE 2020 (Regional Operational Program of the Algarve) and through the Foundation for Science and Technology (FCT), in its national component. M.P. was supported by Portuguese National Funds through FCT/MCTES, under the PhD scholarship SFRH/BD/145321/2019, co-financed by the European Social Fund through the Regional Operational Program of the Alentejo. J.A.R. was supported by Portuguese National Funds through Project ALT20-03-0246-FEDER-000056, “BIOPRO- TOMATE: Bioproteção do tomateiro contra a fusariose—impacto das práticas agronómicas”, under scholarship BI_MESTRE_Uevora_CER_BIOPROTOMATE, co-financed by the European Regional Development Fund through Regional Operational Program Alentejo 2020

Defense Strategies: The Role of Transcription Factors in Tomato–Pathogen Interaction

Tomato, one of the most cultivated and economically important vegetable crops throughout the world, is affected by a panoply of different pathogens that reduce yield and affect product quality. The study of tomato–pathogen system arises as an ideal system for better understanding the molecular mechanisms underlying disease resistance, offering an opportunity of improving yield and quality of the products. Among several genes already identified in tomato response to pathogens, we highlight those encoding the transcription factors (TFs). TFs act as transcriptional activators or repressors of gene expression and are involved in large-scale biological phenomena. They are key regulators of central components of plant innate immune system and basal defense in diverse biological processes, including defense responses to pathogens. Here, we present an overview of recent studies of tomato TFs regarding defense responses to biotic stresses. Hence, we focus on different families of TFs, selected for their abundance, importance, and availability of functionally well-characterized members in response to pathogen attack. Tomato TFs’ roles and possibilities related to their use for engineering pathogen resistance in tomato are presented. With this review, we intend to provide new insights into the regulation of tomato defense mechanisms against invading pathogens in view of plant breeding.This work was funded by the projects “Development of a new virus-based vector to control TSWV in tomato plants” with the references ALT20-03-0145-FEDER-028266 and PTDC/ASP-PLA/28266/2017, and “Control of olive anthracnose through gene silencing and gene expression using a plant virus vector” with the references ALT20-03-0145-FEDER-028263 and PTDC/ASP-PLA/28263/2017, co-financed by the European Union through the European Regional Development Fund, under the ALENTEJO 2020 (Regional Operational Program of the Alentejo), ALGARVE 2020 (Regional Operational Program of the Algarve) and through the Foundation for Science and Technology (FCT), in its national component. M.P. was supported by Portuguese National Funds through FCT/MCTES, under the PhD scholarship SFRH/BD/145321/2019, co-financed by the European Social Fund through the Regional Operational Program of the Alentejo. This work was also supported by National Funds through FCT under the Project UIDB/05183/2020

Metagenomic Assessment Unravels Fungal Microbiota Associated to Grapevine Trunk Diseases

Grapevine trunk diseases (GTDs) are among the most important problems that affect the longevity and productivity of vineyards in all the major growing regions of the world. They are slow-progression diseases caused by several wood-inhabiting fungi with similar life cycles and epidemiology. The simultaneous presence of multiple trunk pathogens in a single plant together with the inconsistent GTDs symptoms expression, their isolation in asymptomatic plants, and the absence of effective treatments make these diseases extremely complex to identify and eradicate. Aiming to gain a better knowledge of GTDs and search sustainable alternatives to limit their development, the present work studied the fungal community structure associated with GTDs symptomatic and asymptomatic grapevines, following a metagenomic approach. Two important cultivars from the Alentejo region with different levels of susceptibility to GTDs were selected, namely, ‘Alicante Bouschet’ and ‘Trincadeira’. Deep sequencing of fungal-directed ITS1 amplicon led to the detection of 258 taxa, including 10 fungi previously described as responsible for GTDs. Symptomatic plants exhibited a lower abundance of GTDs-associated fungi, although with significantly higher diversity of those pathogens. Our results demonstrated that trunk diseases symptoms are intensified by a set of multiple GTDs-associated fungi on the same plant. The composition of fungal endophytic communities was significantly different according to the symptomatology and it was not affected by the cultivar. This study opens new perspectives in the study of GTDs-associated fungi and their relation to the symptomatology in grapevines.This research was supported by Portuguese National Funds through FCT- MCTES under the PhD scholarship SFRH/BD/145321/2019, attributed to M.P., co-financed by the European Social Fund through the Regional Operational Program of the Alentejo. It was also supported by the project “Control of olive anthracnose through gene silencing and gene expression using a plant virus vector” (ALT20-03-0145-FEDER-028263 and PTDC/ASP-PLA/28263/2017) and by the project “Development of a new virus-based vector to control TSWV in tomato plants” (ALT20-03- 0145-FEDER-028266 and PTDC/ASP-PLA/28266/2017), both projects co-financed by the European Union through the European Regional Development Fund, under the Alentejo 2020, Algarve 2020 and through the FCT, in its national component

A degenerate pair of primers for simultaneous detection of four alpha- and betanecroviruses

The high infection levels due to Olive latent virus 1 (OLV-1), Olive mild mosaic virus (OMMV) (alphanecrovirus) and Tobacco necrosis virus D (TNV-D) (betanecrovirus) in Portuguese olive orchards prompted us to develop a rapid PCR-based assay for the simultaneous detection of these viruses aimed at the sanitary selection and marketing of plant material in compliance with European Union regulations. A pair of degenerate oligonucleotide primers, parRdRp5 and parCoat3 was designed based on conserved regions located in the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of these viruses and one other alphanecrovirus, Tobacco necrosis virus A. Its use in RT-PCR assays generated a product of ca. 2000 bp for the 4 viral species tested. These primers were compared with virus specific primers in multiplex RT-PCR, and identical results were obtained. Its application to dsRNA extracted from 54 olive field growing trees originated the expected ca. 2000 bp amplicon in 17 trees. The virus identity was determined by sequencing the cloned RT-PCR products. No TNV-A was found. The RT-PCR assay using the degenerate primers described in this study were shown to be reliable in detecting any of the above-mentioned alpha- and betanecroviruses, and it is as sensitive as that which uses virus specific primers in multiplex assays. Therefore, this assay is well suited for the rapid screen of virus-free plant material in selection and improvement crop programmes. Additionally, it has the potential to reveal virus diversity and the presence of new viruses, provided the RT-PCR generated amplicon is further sequenced

CRISPR/Cas13 for the Control of Plant Viruses

Plant viruses are one of the main threats to crops and food security worldwide, being very difficult to monitor and control. Since there are no effective chemical products against plant viruses, using virus-resistant plants is often the only option. Many promising strategies have been developed to engineer virus resistance however, several setbacks have hampered their utility in agriculture. In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins are players that are involved in the adaptative immune systems against viruses. They have been found and studied over the past decade and can be used as a rapid and simplified technology to develop resistance to viruses overcoming a lot of challenges faced by other techniques. The first CRISPR/Cas system studied was very useful for DNA targeting, but more recently identified types, using Cas13 proteins, that can specifically cleave single-stranded RNA in eukaryotic cells, have enabled a host of new opportunities, especially since most plant viruses have RNA genomes. This chapter aims to bring together the most up-to-date information about CRISPR/Cas13 systems to control plant viruses. We also discuss the limitations and future challenges of the use of CRISPR/Cas13 to produce virus-resistant plants for sustainable agriculture