Recent developments in plant-downy mildew interactions.

Downy mildews are obligate oomycete pathogens that attack a wide range of plants and can cause significant economic impacts on commercial crops and ornamental plants. Traditionally, downy mildew disease control relied on an integrated strategies, that incorporate cultural practices, deployment of resistant cultivars, crop rotation, application of contact and systemic pesticides, and biopesticides. Recent advances in genomics provided data that significantly advanced understanding of downy mildew evolution, taxonomy and classification. In addition, downy mildew genomics also revealed that these obligate oomycetes have reduced numbers of virulence factor genes in comparison to hemibiotrophic and necrotrophic oomycetes. However, downy mildews do deploy significant arrays of virulence proteins, including so-called RXLR proteins that promote virulence or are recognized as avirulence factors. Pathogenomics are being applied to downy mildew population studies to determine the genetic diversity within the downy mildew populations and manage disease by selection of appropriate varieties and management strategies. Genome editing technologies have been used to manipulate host disease susceptibility genes in different plants including grapevine and sweet basil and thereby provide new soucres of resistance genes against downy mildews. Previously, it has proved difficult to transform and manipulate downy mildews because of their obligate lifestyle. However, recent exploitation of RNA interference machinery through Host-Induced Gene Silencing (HIGS) and Spray-Induced Gene Silencing (SIGS) indicate that functional genomics in downy mildews is now possible. Altogether, these breakthrough technologies and attendant fundamental understanding will advance our ability to mitigate downy mildew diseases.

Control of Sclerotinia sclerotiorum via an RNA interference (RNAi)-mediated targeting of SsPac1 and SsSmk1.

MAIN CONCLUSION: The study evaluates the potential of Spray-Induced Gene Silencing and Host-Induced Gene Silencing for sustainable crop protection against the broad-spectrum necrotrophic fungus Sclerotinia sclerotiorum. Sclerotinia sclerotiorum (Lib.) de Bary, an aggressive ascomycete fungus causes white rot or cottony rot on a broad range of crops including Brassica juncea. The lack of sustainable control measures has necessitated biotechnological interventions such as RNA interference (RNAi) for effective pathogen control. Here we adopted two RNAi-based strategies-Spray-Induced Gene Silencing (SIGS) and Host-Induced Gene Silencing (HIGS) to control S. sclerotiorum. SIGS was successful in controlling white rot on Nicotiana benthamiana and B. juncea by targeting SsPac1, a pH-responsive transcription factor and SsSmk1, a MAP kinase involved in fungal development and pathogenesis. Topical application of dsRNA targeting SsPac1 and SsSmk1 delayed infection initiation and progression on B. juncea. Further, altered hyphal morphology and reduced radial growth were also observed following dsRNA application. We also explored the impact of stable dsRNA expression in A. thaliana against S. sclerotiorum. In this report, we highlight the utility of RNAi as a biofungicide and a tool for preliminary functional genomics.

RAS signalling genes can be used as host-induced gene silencing targets to control fungal diseases caused by Sclerotinia sclerotiorum and Botrytis cinerea.

Sclerotinia sclerotiorum causes white mold (also called stem rot, Sclerotinia blight, etc.) in many economically important plants. It is a notorious soilborne fungal pathogen due to its wide host range and ability to survive in soil for long periods of time as sclerotia. Although host-induced gene silencing (HIGS) was recently demonstrated to be an effective method for controlling white mold, limited gene targets are available. Here, using a forward genetics approach, we identified a RAS-GTPase activating protein, SsGAP1, which plays essential roles in sclerotia formation, compound appressoria production and virulence. In parallel, as revealed by our knockout analysis, the SsGAP1 ortholog in Botrytis cinerea, BcGAP1, plays similar roles in fungal development and virulence. By knocking down SsRAS1 and SsRAS2, we also revealed that both SsRAS1 and SsRAS2 are required for vegetative growth, sclerotia development, compound appressoria production and virulence in S. sclerotiorum. Due to the major roles these RAS signalling components play in Sclerotiniaceae biology, they can be used as HIGS targets to control diseases caused by both S. sclerotiorum and B. cinerea. Indeed, when we introduced HIGS constructs targeting SsGAP1, SsRAS1 and SsRAS2 in Nicotiana benthamiana and Arabidopsis thaliana, we observed reduced virulence. Taken together, our forward genetics gene discovery pipeline in S. sclerotiorum is highly effective in identifying novel HIGS targets to control S. sclerotiorum and B. cinerea.

Gene silencing in broomrapes and other parasitic plants of the Orobanchaceae family: mechanisms, considerations, and future directions.

Holoparasites of the Orobanchaceae family are devastating pests causing severe damage to many crop species and are nearly impossible to control with conventional methods. During past decades RNA interference (RNAi) has been seen as a promising approach to control various crop pests. The exchange of small RNAs (sRNAs) between crops and parasitic plants has been documented indicating a potential for the development of methods to protect them via the delivery of the sRNAs to parasites, called host-induced gene silencing (HIGS). Here we describe various approaches used for gene silencing in plants and suggest solutions to improve the long-distance movement of the silencing triggers to elevate the HIGS efficiency in parasitic plants. We also investigate the important biological processes during parasites life cycle with a focus on broomrape species, providing several appropriate target genes that can be used in, especially, multiplex gene silencing experiments. We also touch on how the application of nanoparticles can improve the stability and delivery of the silencing triggers, highlighting its potential for parasitic plants control. Finally, suggestions for further research and possible directions for RNAi in parasitic plants are provided.

Challenges and Opportunities Arising from Host-Botrytis cinerea Interactions to Outline Novel and Sustainable Control Strategies: The Key Role of RNA Interference.

The necrotrophic plant pathogenic fungus Botrytis cinerea (Pers., 1794), the causative agent of gray mold disease, causes significant losses in agricultural production. Control of this fungal pathogen is quite difficult due to its wide host range and environmental persistence. Currently, the management of the disease is still mainly based on chemicals, which can have harmful effects not only on the environment and on human health but also because they favor the development of strains resistant to fungicides. The flexibility and plasticity of B. cinerea in challenging plant defense mechanisms and its ability to evolve strategies to escape chemicals require the development of new control strategies for successful disease management. In this review, some aspects of the host-pathogen interactions from which novel and sustainable control strategies could be developed (e.g., signaling pathways, molecules involved in plant immune mechanisms, hormones, post-transcriptional gene silencing) were analyzed. New biotechnological tools based on the use of RNA interference (RNAi) are emerging in the crop protection scenario as versatile, sustainable, effective, and environmentally friendly alternatives to the use of chemicals. RNAi-based fungicides are expected to be approved soon, although they will face several challenges before reaching the market.

RNA interference: a promising biotechnological approach to combat plant pathogens, mechanism and future prospects.

Plant pathogens and other biological pests represent significant obstacles to crop Protection worldwide. Even though there are many effective conventional methods for controlling plant diseases, new methods that are also effective, environmentally safe, and cost-effective are required. While plant breeding has traditionally been used to manipulate the plant genome to develop resistant cultivars for controlling plant diseases, the emergence of genetic engineering has introduced a completely new approach to render plants resistant to bacteria, nematodes, fungi, and viruses. The RNA interference (RNAi) approach has recently emerged as a potentially useful tool for mitigating the inherent risks associated with the development of conventional transgenics. These risks include the use of specific transgenes, gene control sequences, or marker genes. Utilizing RNAi to silence certain genes is a promising solution to this dilemma as disease-resistant transgenic plants can be generated within a legislative structure. Recent investigations have shown that using target double stranded RNAs via an effective vector system can produce significant silencing effects. Both dsRNA-containing crop sprays and transgenic plants carrying RNAi vectors have proven effective in controlling plant diseases that threaten commercially significant crop species. This article discusses the methods and applications of the most recent RNAi technology for reducing plant diseases to ensure sustainable agricultural yields.

RNA Interference Past and Future Applications in Plants.

Antisense RNA was observed to elicit plant disease resistance and post-translational gene silencing (PTGS). The universal mechanism of RNA interference (RNAi) was shown to be induced by double-stranded RNA (dsRNA), an intermediate produced during virus replication. Plant viruses with a single-stranded positive-sense RNA genome have been instrumental in the discovery and characterization of systemic RNA silencing and suppression. An increasing number of applications for RNA silencing have emerged involving the exogenous application of dsRNA through spray-induced gene silencing (SIGS) that provides specificity and environmentally friendly options for crop protection and improvement.

RNAi Technology: A New Path for the Research and Management of Obligate Biotrophic Phytopathogenic Fungi.

Powdery mildew and rust fungi are major agricultural problems affecting many economically important crops and causing significant yield losses. These fungi are obligate biotrophic parasites that are completely dependent on their hosts for growth and reproduction. Biotrophy in these fungi is determined by the presence of haustoria, specialized fungal cells that are responsible for nutrient uptake and molecular dialogue with the host, a fact that undoubtedly complicates their study under laboratory conditions, especially in terms of genetic manipulation. RNA interference (RNAi) is the biological process of suppressing the expression of a target gene through double-stranded RNA that induces mRNA degradation. RNAi technology has revolutionized the study of these obligate biotrophic fungi by enabling the analysis of gene function in these fungal. More importantly, RNAi technology has opened new perspectives for the management of powdery mildew and rust diseases, first through the stable expression of RNAi constructs in transgenic plants and, more recently, through the non-transgenic approach called spray-induced gene silencing (SIGS). In this review, the impact of RNAi technology on the research and management of powdery mildew and rust fungi will be addressed.

SsCak1 Regulates Growth and Pathogenicity in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating fungal pathogen that causes severe crop losses worldwide. It is of vital importance to understand its pathogenic mechanism for disease control. Through a forward genetic screen combined with next-generation sequencing, a putative protein kinase, SsCak1, was found to be involved in the growth and pathogenicity of S. sclerotiorum. Knockout and complementation experiments confirmed that deletions in SsCak1 caused defects in mycelium and sclerotia development, as well as appressoria formation and host penetration, leading to complete loss of virulence. These findings suggest that SsCak1 is essential for the growth, development, and pathogenicity of S. sclerotiorum. Therefore, SsCak1 could serve as a potential target for the control of S. sclerotiorum infection through host-induced gene silencing (HIGS), which could increase crop resistance to the pathogen.

Silencing a Chitinase Gene, PstChia1, Reduces Virulence of Puccinia striiformis f. sp. tritici.

Chitin is the main component of fungal cell walls, which can be recognized by pattern recognition receptors (PRRs) as pathogen-associated molecular patterns (PAMP). Chitinase in filamentous fungi has been reported to degrade immunogenic chitin oligomers, thereby preventing chitin-induced immune activation. In this study, we identified the chitinase families in 10 fungal genomes. A total of 131 chitinase genes were identified. Among the chitinase families, 16 chitinase genes from Puccinia striiformis f. sp. tritici (Pst) were identified, and the expression of PstChia1 was the highest during Pst infection. Further studies indicated that PstChia1 is highly induced during the early stages of the interaction of wheat and Pst and has chitinase enzyme activity. The silencing of PstChia1 revealed that PstChia1 limited the growth and reduced the virulence of Pst. The expression level of TaPR1 and TaPR2 was induced in PstChia1 knockdown plants, suggesting that PstChia1 is involved in regulating wheat resistance to Pst. Our data suggest that PstChia1 contributes to pathogenicity by interfering with plant immunity and regulating the growth of Pst.

RNA-Based Control of Fungal Pathogens in Plants.

Our duty to conserve global natural ecosystems is increasingly in conflict with our need to feed an expanding population. The use of conventional pesticides not only damages the environment and vulnerable biodiversity but can also still fail to prevent crop losses of 20-40% due to pests and pathogens. There is a growing call for more ecologically sustainable pathogen control measures. RNA-based biopesticides offer an eco-friendly alternative to the use of conventional fungicides for crop protection. The genetic modification (GM) of crops remains controversial in many countries, though expression of transgenes inducing pathogen-specific RNA interference (RNAi) has been proven effective against many agronomically important fungal pathogens. The topical application of pathogen-specific RNAi-inducing sprays is a more responsive, GM-free approach to conventional RNAi transgene-based crop protection. The specific targeting of essential pathogen genes, the development of RNAi-nanoparticle carrier spray formulations, and the possible structural modifications to the RNA molecules themselves are crucial to the success of this novel technology. Here, we outline the current understanding of gene silencing pathways in plants and fungi and summarize the pioneering and recent work exploring RNA-based biopesticides for crop protection against fungal pathogens, with a focus on spray-induced gene silencing (SIGS). Further, we discuss factors that could affect the success of RNA-based control strategies, including RNA uptake, stability, amplification, and movement within and between the plant host and pathogen, as well as the cost and design of RNA pesticides.

Reduction of Rhizoctonia cerealis Infection on Wheat Through Host- and Spray-Induced Gene Silencing of an Orphan Secreted Gene.

Rhizoctonia cerealis is a soilborne fungus that can cause sharp eyespot in wheat, resulting in massive yield losses found in many countries. Due to the lack of resistant cultivars, fungicides have been widely used to control this pathogen. However, chemical control is not environmentally friendly and is costly. Meanwhile, the lack of genetic transformation tools has hindered the functional characterization of virulence genes. In this study, we attempted to characterize the function of virulence genes by two transient methods, host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), which use RNA interference to suppress the pathogenic development. We identified ten secretory orphan genes from the genome. After silencing these ten genes, only the RcOSP1 knocked-down plant significantly inhibited the growth of R. cerealis. We then described RcOSP1 as an effector that could impair wheat biological processes and suppress pathogen-associated molecular pattern-triggered immunity in the infection process. These findings confirm that HIGS and SIGS can be practical tools for researching R. cerealis virulence genes. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

New Insights on the Integrated Management of Plant Diseases by RNA Strategies: Mycoviruses and RNA Interference.

RNA-based strategies for plant disease management offer an attractive alternative to agrochemicals that negatively impact human and ecosystem health and lead to pathogen resistance. There has been recent interest in using mycoviruses for fungal disease control after it was discovered that some cause hypovirulence in fungal pathogens, which refers to a decline in the ability of a pathogen to cause disease. Cryphonectria parasitica, the causal agent of chestnut blight, has set an ideal model of management through the release of hypovirulent strains. However, mycovirus-based management of plant diseases is still restricted by limited approaches to search for viruses causing hypovirulence and the lack of protocols allowing effective and systemic virus infection in pathogens. RNA interference (RNAi), the eukaryotic cell system that recognizes RNA sequences and specifically degrades them, represents a promising. RNA-based disease management method. The natural occurrence of cross-kingdom RNAi provides a basis for host-induced gene silencing, while the ability of most pathogens to uptake exogenous small RNAs enables the use of spray-induced gene silencing techniques. This review describes the mechanisms behind and the potential of two RNA-based strategies, mycoviruses and RNAi, for plant disease management. Successful applications are discussed, as well as the research gaps and limitations that remain to be addressed.

RNA Interference: Promising Approach to Combat Plant Viruses.

Plant viruses are devastating plant pathogens that severely affect crop yield and quality. Plants have developed multiple lines of defense systems to combat viral infection. Gene silencing/RNA interference is the key defense system in plants that inhibits the virulence and multiplication of pathogens. The general mechanism of RNAi involves (i) the transcription and cleavage of dsRNA into small RNA molecules, such as microRNA (miRNA), or small interfering RNA (siRNA), (ii) the loading of siRNA/miRNA into an RNA Induced Silencing Complex (RISC), (iii) complementary base pairing between siRNA/miRNA with a targeted gene, and (iv) the cleavage or repression of a target gene with an Argonaute (AGO) protein. This natural RNAi pathway could introduce transgenes targeting various viral genes to induce gene silencing. Different RNAi pathways are reported for the artificial silencing of viral genes. These include Host-Induced Gene Silencing (HIGS), Virus-Induced Gene Silencing (VIGS), and Spray-Induced Gene Silencing (SIGS). There are significant limitations in HIGS and VIGS technology, such as lengthy and time-consuming processes, off-target effects, and public concerns regarding genetically modified (GM) transgenic plants. Here, we provide in-depth knowledge regarding SIGS, which efficiently provides RNAi resistance development against targeted genes without the need for GM transgenic plants. We give an overview of the defense system of plants against viral infection, including a detailed mechanism of RNAi, small RNA molecules and their types, and various kinds of RNAi pathways. This review will describe how RNA interference provides the antiviral defense, recent improvements, and their limitations.

Host-induced gene silencing - mechanisms and applications.

Host-induced gene silencing (HIGS) technology has emerged as a powerful alternative to chemical treatments for protecting plants from pathogens or pests. More than 170 HIGS studies have been published so far, and HIGS products have been launched. First, we discuss the strengths and limitations of this technology in a pathosystem-specific context. Next, we highlight the requirement for fundamental knowledge on the molecular mechanisms (i.e. uptake, processing and translocation of transgene-expressed double-stranded RNAs) that determine the efficacy and specificity of HIGS. Additionally, we speculate on the contribution of host and target RNA interference machineries, which may be incompatible depending on the lifestyle of the pathogen or pest. Finally, we predict that closing these gaps in knowledge will lead to the development of novel integrative concepts, precise risk assessment and tailor-made HIGS therapy for plant diseases.

An Arabidopsis downy mildew non-RxLR effector suppresses induced plant cell death to promote biotroph infection.

Our understanding of obligate biotrophic pathogens is limited by lack of knowledge concerning the molecular function of virulence factors. We established Arabidopsis host-induced gene silencing (HIGS) to explore gene functions of Hyaloperonospora arabidopsidis, including CYSTEINE-RICH PROTEIN (HaCR)1, a potential secreted effector gene of this obligate biotrophic pathogen. HaCR1 HIGS resulted in H. arabidopsidis-induced local plant cell death and reduced pathogen reproduction. We functionally characterized HaCR1 by ectopic expression in Nicotiana benthamiana. HaCR1 was capable of inhibiting effector-triggered plant cell death. Consistent with this, HaCR1 expression in N. benthamiana led to stronger disease symptoms caused by the hemibiotrophic oomycete pathogen Phytophthora capsici, but reduced disease symptoms caused by the necrotrophic fungal pathogen Botrytis cinerea. Expressing HaCR1 in transgenic Arabidopsis confirmed higher susceptibility to H. arabidopsidis and to the bacterial hemibiotrophic pathogen Pseudomonas syringae. Increased H. arabidopsidis infection was in accordance with reduced PATHOGENESIS RELATED (PR)1 induction. Expression of full-length HaCR1 was required for its function, which was lost if the signal peptide was deleted, suggesting its site of action in the plant apoplast. This study provides phytopathological and molecular evidence for the importance of this widespread, but largely unexplored class of non-RxLR effectors in biotrophic oomycetes.

Recent Progress in Enhancing Fungal Disease Resistance in Ornamental Plants.

Fungal diseases pose a major threat to ornamental plants, with an increasing percentage of pathogen-driven host losses. In ornamental plants, management of the majority of fungal diseases primarily depends upon chemical control methods that are often non-specific. Host basal resistance, which is deficient in many ornamental plants, plays a key role in combating diseases. Despite their economic importance, conventional and molecular breeding approaches in ornamental plants to facilitate disease resistance are lagging, and this is predominantly due to their complex genomes, limited availability of gene pools, and degree of heterozygosity. Although genetic engineering in ornamental plants offers feasible methods to overcome the intrinsic barriers of classical breeding, achievements have mainly been reported only in regard to the modification of floral attributes in ornamentals. The unavailability of transformation protocols and candidate gene resources for several ornamental crops presents an obstacle for tackling the functional studies on disease resistance. Recently, multiomics technologies, in combination with genome editing tools, have provided shortcuts to examine the molecular and genetic regulatory mechanisms underlying fungal disease resistance, ultimately leading to the subsequent advances in the development of novel cultivars with desired fungal disease-resistant traits, in ornamental crops. Although fungal diseases constitute the majority of ornamental plant diseases, a comprehensive overview of this highly important fungal disease resistance seems to be insufficient in the field of ornamental horticulture. Hence, in this review, we highlight the representative mechanisms of the fungal infection-related resistance to pathogens in plants, with a focus on ornamental crops. Recent progress in molecular breeding, genetic engineering strategies, and RNAi technologies, such as HIGS and SIGS for the enhancement of fungal disease resistance in various important ornamental crops, is also described.

Host-induced silencing of the Colletotrichum gloeosporioides conidial morphology 1 gene (CgCOM1) confers resistance against Anthracnose disease in chilli and tomato.

KEY MESSAGE: Host mediated silencing of COM1 gene of Colletotrichum gloeosporioides disables appressorial differentiation and effectively prevents the development of Anthracnose disease in chilli and tomato. Anthracnose disease is caused by the ascomycetes fungal species Colletotrichum, which is responsible for heavy yield losses in chilli and tomato worldwide. Conventionally, harmful pesticides are used to contain anthracnose disease with limited success. In this study, we assessed the potential of Host-Induced Gene Silencing (HIGS) approach to target the Colletotrichum gloeosporioides COM1 (CgCOM1) developmental gene involved in the fungal conidial and appressorium formation, to restrict fungal infection in chilli and tomato fruits. For this study, we have developed stable transgenic lines of chilli and tomato expressing CgCOM1-RNAi construct employing Agrobacterium-mediated transformation. Transgenic plants were characterized by molecular and gene expression analyses. Production of specific CgCOM1 siRNA in transgenic chilli and tomato RNAi lines was confirmed by stem-loop RT-PCR. Fungal challenge assays on leaves and fruits showed that the transgenic lines were resistant to anthracnose disease-causing C. gloeosporioides in comparison to wild type and empty-vector control plants. RT-qPCR analyses in transgenic lines revealed extremely low abundance of CgCOM1 transcripts in the C. gloeosporioides infected tissues, indicating near complete silencing of CgCOM1 gene expression in the pathogen. Microscopic examination of the Cg-challenged leaves of chilli-CgCOM1i lines revealed highly suppressed conidial germination, germ tube development, appressoria formation and mycelial growth of C. gloeosporioides, resulting in reduced infection of plant tissues. These results demonstrated highly efficient use of HIGS in silencing the expression of essential fungal developmental genes to inhibit the growth of pathogenic fungi, thus providing a highly precise approach to arrest the spread of disease.

Host-induced gene silencing of the acetolactate synthases VdILV2 and VdILV6 confers resistance to Verticillium wilt in cotton (Gossypium hirsutum L.).

Cotton Verticillium wilt caused by Verticillium dahliae (V. dahliae) is one of the most destructive fungal diseases and is difficult to control. However, resistant germplasm resources are scarce in cotton. Many studies have shown that host-induced gene silencing (HIGS) is a practical and effective technology in crop disease prevention by silencing virulence genes of pathogens. Acetolactate synthase (ALS) contains a catalytic subunit ILV2 and a regulatory subunit ILV6, which catalyzes the first common step reaction in branched-chain amino acid (BCAA) biosynthesis. We identified two acetolactate synthases, VdILV2 and VdILV6, which are homologs of ILV2 and ILV6, respectively, in Magnaporthe oryzae. To characterize the function of VdILV2 and VdILV6 in V. dahliae, we suppressed their expression in the strong pathogenic isolate Vd991 by using HIGS technology. VdILV2- or VdILV6-silenced V. dahliae had a dramatic reduction in pathogenicity. The results indicated that VdILV2 and VdILV6 are involved in the pathogenicity of V. dahliae. HIGS of VdILV2 or VdILV6 provides a novel fungicide target and an effective control to resist Verticillium wilt caused by V. dahliae.

Growing pains: addressing the pitfalls of plant extracellular vesicle research.

Extracellular vesicles (EVs) are small, membrane-enclosed compartments that mediate the intercellular transport of proteins and small RNAs. In plants, EVs are thought to play a prominent role in immune responses and are being championed as the long-sought-after mechanism for host-induced gene silencing. However, parallel research on mammalian EVs is raising concerns about potential pitfalls faced by all EV researchers that will need to be addressed in order to convincingly establish that EVs are the primary mediators of small RNA transfer between organisms. Here we discuss these pitfalls in the context of plant EV research, with a focus on experimental approaches required to distinguish bona fide EV cargo from merely co-purifying contaminants.