Plant Viruses: From Targets to Tools for CRISPR

Plant viruses cause devastating diseases in many agriculture systems, being a serious threat for the provision of adequate nourishment to a continuous growing population. At the present, there are no chemical products that directly target the viruses, and their control rely mainly on preventive sanitary measures to reduce viral infections that, although important, have proved to be far from enough. The current most effective and sustainable solution is the use of virus-resistant varieties, but which require too much work and time to obtain. In the recent years, the versatile gene editing technology known as CRISPR/Cas has simplified the engineering of crops and has successfully been used for the development of viral resistant plants. CRISPR stands for ‘clustered regularly interspaced short palindromic repeats’ and CRISPR-associated (Cas) proteins, and is based on a natural adaptive immune system that most archaeal and some bacterial species present to defend themselves against invading bacteriophages. Plant viral resistance using CRISPR/Cas technology can been achieved either through manipulation of plant genome (plant-mediated resistance), by mutating host factors required for viral infection; or through manipulation of virus genome (virus-mediated resistance), for which CRISPR/Cas systems must specifically target and cleave viral DNA or RNA. Viruses present an efficient machinery and comprehensive genome structure and, in a different, beneficial perspective, they have been used as biotechnological tools in several areas such as medicine, materials industry, and agriculture with several purposes. Due to all this potential, it is not surprising that viruses have also been used as vectors for CRISPR technology; namely, to deliver CRISPR components into plants, a crucial step for the success of CRISPR technology. Here we discuss the basic principles of CRISPR/Cas technology, with a special focus on the advances of CRISPR/Cas to engineer plant resistance against DNA and RNA viruses. We also describe several strategies for the delivery of these systems into plant cells, focusing on the advantages and disadvantages of the use of plant viruses as vectors. We conclude by discussing some of the constrains faced by the application of CRISPR/Cas technology in agriculture and future prospects

High throughput sequencing unravels tomato- pathogen interactions towards a sustainable plant breeding

Tomato (Solanum lycopersicum) is one of the most economically important vegetables throughout the world. It is one of the best studied cultivated dicotyledonous plants, often used as a model system for plant research into classical genetics, cytogenetics, molecular genetics, and molecular biology. Tomato plants are affected by different pathogens such as viruses, viroids, fungi, oomycetes, bacteria, and nematodes, that reduce yield and affect product quality. The study of tomato as a plant-pathogen system helps to accelerate the discovery and understanding of the molecular mechanisms underlying disease resistance and offers the opportunity of improving the yield and quality of their edible products. The use of functional genomics has contributed to this purpose through both traditional and recently developed techniques, that allow the identification of plant key functional genes in susceptible and resistant responses, and the understanding of the molecular basis of compatible interactions during pathogen attack. Nextgeneration sequencing technologies (NGS), which produce massive quantities of sequencing data, have greatly accelerated research in biological sciences and offer great opportunities to better understand the molecular networks of plant–pathogen interactions. In this review, we summarize important research that used high-throughput RNA-seq technology to obtain transcriptome changes in tomato plants in response to a wide range of pathogens such as viruses, fungi, bacteria, oomycetes, and nematodes. These findings will facilitate genetic engineering efforts to incorporate new sources of resistance in tomato for protection against pathogens and are of major importance for sustainable plant-disease management, namely the ones relying on the plant’s innate immune mechanisms in view of plant breeding

The dual role of Plant Viruses in CRISPR

Plant viruses cause devastating diseases in many agriculture systems, being a serious threat for the provision of adequate nourishment to a continuous growing population. At the present there are no chemical products that directly target the viruses, and their control rely mainly on preventive sanitary measures to reduce viral infections that, although important, have proved to be far from enough. The current most effective and sustainable solution is the use of virusresistant varieties, which require too much work and time to obtain. In the recent years, the versatile gene editing technology known as CRISPR/Cas has simplified the engineering of crops and has successfully been used for the development of viral resistant plants. CRISPR stands for Clustered regularly interspaced short palindromic repeats and CRISPR-associated (Cas) proteins, and is based on a natural adaptive immune system that most archaeal and some bacterial species present to defend themselves against invading bacteriophages. Plant viral resistance using CRISPR/Cas technology has been achieved either through manipulation of plant genome (plant-mediated resistance), by mutating host factors required for viral infection, or through manipulation of virus genome (virus-mediated resistance), for which CRISPR/Cas systems must specifically target and cleave viral DNA or RNA. Viruses present an efficient machinery and comprehensive genome structure and, in a different perspective, they have been used as biotechnological tools in several areas such as medicine, materials industry and agriculture with several purposes. Due to all this potential, it is not surprising that viruses have also been used as vectors for CRISPR technology, namely to deliver CRISPR components into plants, a crucial step for the success of CRISPR technology. Here we discuss the basic principles of CRISPR/Cas technology, with a special focus on the advances of CRISPR/Cas to engineer plant resistance against DNA and RNA viruses. We also describe several strategies for the delivery of these systems into plant cells, focusing on the advantages and disadvantages of the use of plant viruses as vectors. We conclude by discussing the constrains faced by the application of CRISPR/Cas technology in agriculture and future prospects

Plant-Pathogen Interaction

Plant diseases result in severe losses to natural plant systems, and also cause problems for economics and production in agricultural systems. While many biotic constraints are well known and confronted with variable success, the occurrence of emerging pathogens and the progressive incidence of novel virulent strains, races, or pathotypes is evident. Plant disease management faces challenges due to the increasing incidence of emergent diseases, with a consequent decrease in the production potential of agriculture. Furthermore, the deteriorating ecology of agro-ecosystems and the depletion of natural resources, together with an increased risk of disease epidemics resulting from agricultural intensification and monocultures, should be taken into account. Moreover, the practicability of some of the currently available plant protection measures is questionable. The UE directories for commercialization withdrawal of several chemical substances used for pest and disease control, and the new rules for reducing agricultural greenhouse gas emissions contained in the 2015 Paris Agreement of the United Nations Framework Convention on Climate Change, bring new challenges to agricultural production

Metagenomic analysis of fungal microbiota associated to grapevine trunk diseases in Alentejo region

Grapevine trunk diseases (GTDs) are considered among the most important problems affecting the longevity and productivity of vineyards in all the major growing regions of the world, causing important economic losses. They are caused by wood inhabiting fungi, namely by 133 species belonging to 9 families and 34 genera, with similar life cycles and epidemiology. Until now, no effective treatments are known. Aiming to gain a better knowledge of these diseases and search alternatives to limit their development, the present work intended to molecularly identify GTDs- associated fungi, grapevine endophytic community and fungi with antagonist ability against GTDs. For this study, two important cultivars from the Alentejo region were selected, ‘Alicante Bouschet’ and ‘Trincadeira’, which demonstrate different levels of susceptibility to GTDs. Samples consisted of cuttings from plants with and without trunk diseases symptoms from both cultivars in a vineyard located in this region. Total DNA was extracted from cortical scrapings and sequenced using a metagenomic approach based on next generation sequence analysis. Deep sequencing of fungal-directed ITS1 and ITS2 amplicons led to the detection of 215 taxa in grapevine fungal microbiota, with nine fungi previously described as responsible for GTDs. Unexpectedly, symptomatic plants showed a lower relative abundance of GTDs-associated fungi and a higher relative abundance of possible antagonist fungi, in opposition to what was obtained in asymptomatic plants in both cultivars. Nevertheless, symptomatic plants showed greater diversity of GTDs phytopathogenic fungi when compared to asymptomatic plants. These facts corroborate previous reports referring that trunk diseases symptoms are intensified by a set of several associated fungi on the same plant. Some fungal species with biological antagonist characteristics were also identified but their role in GTDs still need further investigation. This study allowed a deeper knowledge of grapevine fungal communities of the selected cultivars and updated the information on the abundance and diversity of GTDs associated fungi and their relationship with the symptomatology in plants. Additional studies are still required to better understand plant-pathogen interactions and contribute to the mitigation and control of GTDs in the Alentejo region

A Bipartite Geminivirus with a Highly Divergent Genomic Organization Identified in Olive Trees May Represent a Novel Evolutionary Direction in the Family Geminiviridae

Olea europaea Geminivirus (OEGV) was recently identified in olive in Italy through HTS. In this work, we used HTS to show the presence of an OEGV isolate in Portuguese olive trees and suggest the evolution direction of OEGV. The bipartite genome (DNA-A and DNA-B) of the OEGV-PT is similar to Old World begomoviruses in length, but it lacks a pre-coat protein (AV2), which is a typical feature of NewWorld begomoviruses (NW). DNA-A genome organization is closer to NW, containing four ORFs; three in complementary-sense AC1/Rep, AC2/TrAP, AC3/REn and one in virion-sense AV1/CP, but no AC4, typical of begomoviruses. DNA-B comprises two ORFs; MP in virion sense with higher similarity to the tyrosine phosphorylation site of NW, but in opposite sense to begomoviruses; BC1, with no known conserved domains in the complementary sense and no NSP typical of bipartite begomoviruses. Our results show that OEGV presents the longest common region among the begomoviruses, and the TATA box and four replication-associated iterons in a completely new arrangement. We propose two new putative conserved regions for the geminiviruses CP. Lastly, we highlight unique features that may represent a new evolutionary direction for geminiviruses and suggest that OEGV-PT evolution may have occurred from an ancient OW monopartite Begomovirus that lost V2 and C4, gaining functions on cell-to-cell movement by acquiring a DNA-B component

Reuse of Pretreated Agro-Industrial Wastewaters for Hydroponic Production of Lettuce

The utilization of agro-industrial wastewaters (AIWWs), pretreated by immediate one-step lime precipitation + natural carbonation, as a nutritive solution for the hydroponic production of lettuce was evaluated. The AIWWs studied were olive mill wastewater (OMW), winery wastewater (WW), and cheese whey wastewater (CWW). Lettuces (Lactuca sativa L. var. crispa) were grown in a closed nutrient film technique hydroponic system, using the pretreated AIWWs (OMW-T, WW-T, and CWW-T) and a control nutrient solution (CNS). The growth and sensory analysis of lettuces and the environmental parameters of effluents after hydroponics were evaluated. The average number of lettuce leaves obtained with nutrient solutions prepared with AIWW-T was lower than that from CNS, but the highest lettuce chlorophyll content was attained with CWW-T, which also presented the best grow results. In general, sensory analysis did not show significant differences from the lettuces grown in the different pretreated AIWWs and CNS. As for the environmental parameters of the effluents from hydroponics, according to the Portuguese legislation, only the chemical oxygen demand of the OMW-T and WW-T presented slightly higher values than that of the environmental limit values for discharge in surface waters, showing the feasibility of using pretreated agro-industrial effluents in hydroponic lettuce cultivation, while obtaining a treated effluent, in a circular economy perspective.This research was funded by NETA project, New Strategies in Wastewater Treatment (POCI-01-0247-FEDER-046959), through PORTUGAL2020, by Carbo2Soil project (PRR-C05-i03-I000030), through Plano de Recuperação e Resiliência (PRR), and by Fundação para a Ciência e a Tecnologia, FCT, project UIDB/00195/2020, Ph.D. grant 2020.04822.BD awarded to A. Afonso, and contract awarded to A. Fernandes.info:eu-repo/semantics/publishedVersio

Detection and Quantification of Fusarium spp. (F. oxysporum, F. verticillioides, F. graminearum) and Magnaporthiopsis maydis in Maize Using Real-Time PCR Targeting the ITS Region

Fusarium spp. and Magnaporthiopsis maydis are soil-inhabiting fungi and respectively the causal agents of fusarium ear rot and late wilt, two important diseases that can affect maize, one of the most important cereal crops worldwide. Here, we present two sensitive real-time PCR TaqMan MGB (Minor Groove Binder) assays that detect and discriminate several Fusarium spp. (F. oxysporum, F. verticillioides, and F. graminearum) from M. maydis. The method is based on selective real-time qPCR amplification of the internal transcribed spacer (ITS) region and allows the quantification of the fungi. The applicability of this newly developed TaqMan methodology was demonstrated in a field experiment through the screening of potentially infected maize roots, revealing a high specificity and proving to be a suitable tool to ascertain Fusarium spp. and M. maydis infection in maize. Its high sensitivity makes it very efficient for the early diagnosis of the diseases and also for certification purposes. Thus, qPCR through the use of TaqMan probes is here proposed as a promising tool for specific identification and quantification of these soil-borne fungal pathogens known to cause disease on a large number of crops

Tomato Transcription Factors Regulate Defence Response Against Biotic Stresses

Tomato is one of the most economically important vegetable crops throughout the world. However, it is affected by a panoply of different pathogens that reduce yield and affect product quality, causing symptoms including wilts, leaf spots/blights, fruit spots and rots. Tomato diseases are mainly caused by fungi, but also by oomycetes, bacteria, viruses, viroids and nematodes. The study of plant-pathogen system in tomato 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 that have been identified in tomato response to pathogens, we highlight those encoding the transcription factors (TFs). TFs are considered central components of plant innate immune system and basal defence in diverse biological processes. They act through sequence-specific interactions with cis-regulatory DNA elements in the promoters of genes and are key regulators of tomato defence response against a wide array of pathogens linked to important diseases, together with a complex cross-talk between different signal transduction pathways. Here we discuss recent studies of tomato TFs regarding defence responses to biotic stresses. Hence, we focus on the identification and role of different families of TFs selected for their abundance, importance, and the availability of functionally well-characterized members in response to pathogen attack. Genes that encode TFs as master regulators of stress-related genes offer extended possibilities related to their use for engineering pathogen resistance in tomato plants, arising as candidates for tomato breeding, taking advantage of the newly emerging molecular techniques applied to plant breeding in the genomics and genome editing era