Plant pathogenic fungi are the largest group of disease-causing agents on crop plants and represent a persistent and significant threat to agriculture worldwide. Conventional approaches based on the use of pesticides raise social concern for the impact on the environment and human health and alternative control methods are urgently needed. The rapid improvement and extensive implementation of RNA interference (RNAi) technology for various model and non-model organisms has provided the initial framework to adapt this post-transcriptional gene silencing technology for the management of fungal pathogens. Recent studies showed that the exogenous application of double-stranded RNA (dsRNA) molecules on plants targeting fungal growth and virulence-related genes provided disease attenuation of pathogens like Botrytis cinerea, Sclerotinia sclerotiorum and Fusarium graminearum in different hosts. Such results highlight that the exogenous RNAi holds great potential for RNAi-mediated plant pathogenic fungal disease control. Production of dsRNA can be possible by using either in-vitro or in-vivo synthesis. In this review, we describe exogenous RNAi involved in plant pathogenic fungi and discuss dsRNA production, formulation, and RNAi delivery methods. Potential challenges that are faced while developing a RNAi strategy for fungal pathogens, such as off-target and epigenetic effects, with their possible solutions are also discussed.
Summary Rapid alkalinization factor (RALF) genes encode for ubiquitous small peptides that stimulate apoplastic alkalinization through interaction with malectin‐like receptor kinase. RALF peptides may act as negative regulators of plant immune response, inhibiting the formation of the signal receptor complex for immune activation. Recently RALF homologues were identified in different fungal pathogen genomes contributing to host infection ability. Here, FaRALF‐33‐like gene expression was evaluated in strawberry fruits inoculated with Colletotrichum acutatum, Botrytis cinerea , or Penicillium expansum after 24 and 48 h post‐infection. To investigate the role of FaRALF‐33‐like in strawberry susceptibility, transient transformation was used to overexpress it in white unripe fruits and silence it in red ripe fruits. Agroinfiltrated fruits were inoculated with C. acutatum and expression, and histological analysis of infection were performed. Silencing of FaRALF‐33‐like expression in C. acutatum ‐inoculated red fruits led to a delay in fruit colonization by the fungal pathogen, and infected tissues showed less penetrated infective hyphae than in wild‐type fruits. In contrast, C. acutatum‐ inoculated white unripe fruits overexpressing the FaRALF‐33‐like gene decreased the ontogenic resistance of these fruits, leading to the appearance of disease symptoms and penetrated subcuticular hyphae, normally absent in white unripe fruits. The different response of transfected strawberry fruits to C. acutatum supports the hypothesis that the FaRALF‐33‐like gene plays an important role in the susceptibility of fruits to the fungal pathogen C. acutatum .
Anthracnose is a severe disease caused by Colletotrichum spp. on several crop species. Fungal infections can occur both in the field and at the post-harvest stage causing severe lesions on fruits and economic losses. Physical treatments and synthetic fungicides have traditionally been the preferred means to control anthracnose adverse effects; however, the urgent need to decrease the use of toxic chemicals led to the investigation of innovative and sustainable protection techniques. Evidence for the efficacy of biological agents and vegetal derivates has been reported; however, their introduction into actual crop protection strategies requires the solutions of several critical issues. Biotechnology-based approaches have also been explored, revealing the opportunity to develop innovative and safe methods for anthracnose management through genome editing and RNA interference technologies. Nevertheless, besides the number of advantages related to their use, e.g., the putative absence of adverse effects due to their high specificity, a number of aspects remain to be clarified to enable their introduction into Integrated Pest Management (IPM) protocols against Colletotrichum spp. disease.
Downy mildew caused by Plasmopara viticola is one of the most devastating diseases of grapevine, attacking all green parts of the plant. The damage is severe when the infection at flowering stage is left uncontrolled. P. viticola management consumes a significant amount of classical pesticides applied in vineyards, requiring efficient and environmentally safe disease management options. Spray-induced gene silencing (SIGS), through the application of exogenous double-stranded RNA (dsRNA), has shown promising results for the management of diseases in crops. Here, we developed and tested the potential of dsRNA targeting P. viticola Dicer-like (DCL) genes for SIGS-based crop protection strategy. The exogenous application of PvDCL1/2 dsRNA, a chimera of PvDCL1 and PvDCL2, highly affected the virulence of P. viticola. The reduced expression level of PvDCL1 and PvDCL2 transcripts in infected leaves, treated with PvDCL1/2 dsRNA, was an indication of an active RNA interference mechanism inside the pathogen to compromise its virulence. Besides the protective property, the PvDCL1/2 dsRNA also exhibited a curative role by reducing the disease progress rate of already established infection. Our data provide a promising future for PvDCL1/2 dsRNA as a new generation of RNA-based resistant plants or RNA-based agrochemical for the management of downy mildew disease in grapevine.
Fragaria × ananassa mannose-binding lectin gene, FaMBL1, is a member of strawberry G-type lectin family involved in strawberry defense. Genome-wide identification of G-type lectin gene family was carried out in F. vesca, where 133 G-lectin genes were found. Their expression profile was retrieved from available databases showing that many G-lectin genes are actively expressed during strawberry development or interaction with pathogens. Hence, stable transgenic strawberry plants overexpressing the FaMBL1 gene were generated to better investigate the role of this gene in defense. Transformed strawberry plants were selected and evaluated for their disease-related phytohormones content and reaction to biotic stresses revealing that jasmonic acid (JA) content decreased in overexpressing lines compared to wild type (WT). Petioles of overexpressing lines inoculated with C. fioriniae had lower disease incidence than WT, and leaves of overexpressing lines challenged by B. cinerea showed remarkably smaller lesion diameters compared to WT and higher chitinase 2-1 (FaChi2-1) gene expression. Our results indicate that FaMBL1 gene plays an important role in strawberry response to fungal diseases caused by C. fioriniae and B. cinerea.
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