ATR2C ala2 from Arabidopsis-infecting downy mildew requires 4 TIR-NLR immune receptors for full recognition.

Arabidopsis Col-0 RPP2A and RPP2B confer recognition of Arabidopsis downy mildew (Hyaloperonospora arabidopsidis [Hpa]) isolate Cala2, but the identity of the recognized ATR2Cala2 effector was unknown. To reveal ATR2Cala2, an F2 population was generated from a cross between Hpa-Cala2 and Hpa-Noks1. We identified ATR2Cala2 as a non-canonical RxLR-type effector that carries a signal peptide, a dEER motif, and WY domains but no RxLR motif. Recognition of ATR2Cala2 and its effector function were verified by biolistic bombardment, ectopic expression and Hpa infection. ATR2Cala2 is recognized in accession Col-0 but not in Ler-0 in which RPP2A and RPP2B are absent. In ATR2Emoy2 and ATR2Noks1 alleles, a frameshift results in an early stop codon. RPP2A and RPP2B are essential for the recognition of ATR2Cala2. Stable and transient expression of ATR2Cala2 under 35S promoter in Arabidopsis and Nicotiana benthamiana enhances disease susceptibility. Two additional Col-0 TIR-NLR (TNL) genes (RPP2C and RPP2D) adjacent to RPP2A and RPP2B are quantitatively required for full resistance to Hpa-Cala2. We compared RPP2 haplotypes in multiple Arabidopsis accessions and showed that all four genes are present in all ATR2Cala2-recognizing accessions.

Mapping of the gene in tomato conferring resistance to root-knot nematodes at high soil temperature.

Root-knot nematodes (RKNs, Meloidogyne spp.) can cause severe yield losses in tomatoes. The Mi-1.2 gene in tomato confers resistance to the Meloidogyne species M. incognita, M. arenaria and M. javanica, which are prevalent in tomato growing areas. However, this resistance breaks down at high soil temperatures (>28°C). Therefore, it is imperative that new resistance sources are identified and incorporated into commercial breeding programmes. We identified a tomato line, MT12, that does not have Mi-1.2 but provides resistance to M. incognita at 32°C soil temperature. An F2 mapping population was generated by crossing the resistant line with a susceptible line, MT17; the segregation ratio showed that the resistance is conferred by a single dominant gene, designated RRKN1 (Resistance to Root-Knot Nematode 1). The RRKN1 gene was mapped using 111 Kompetitive Allele Specific PCR (KASP) markers and characterized. Linkage analysis showed that RRKN1 is located on chromosome 6 and flanking markers placed the locus within a 270 kb interval. These newly developed markers can help pyramiding R-genes and generating new tomato varieties resistant to RKNs at high soil temperatures.

Recent Developments in CRISPR/Cas9 Genome-Editing Technology Related to Plant Disease Resistance and Abiotic Stress Tolerance.

The revolutionary CRISPR/Cas9 genome-editing technology has emerged as a powerful tool for plant improvement, offering unprecedented precision and efficiency in making targeted gene modifications. This powerful and practical approach to genome editing offers tremendous opportunities for crop improvement, surpassing the capabilities of conventional breeding techniques. This article provides an overview of recent advancements and challenges associated with the application of CRISPR/Cas9 in plant improvement. The potential of CRISPR/Cas9 in terms of developing crops with enhanced resistance to biotic and abiotic stresses is highlighted, with examples of genes edited to confer disease resistance, drought tolerance, salt tolerance, and cold tolerance. Here, we also discuss the importance of off-target effects and the efforts made to mitigate them, including the use of shorter single-guide RNAs and dual Cas9 nickases. Furthermore, alternative delivery methods, such as protein- and RNA-based approaches, are explored, and they could potentially avoid the integration of foreign DNA into the plant genome, thus alleviating concerns related to genetically modified organisms (GMOs). We emphasize the significance of CRISPR/Cas9 in accelerating crop breeding processes, reducing editing time and costs, and enabling the introduction of desired traits at the nucleotide level. As the field of genome editing continues to evolve, it is anticipated that CRISPR/Cas9 will remain a prominent tool for crop improvement, disease resistance, and adaptation to challenging environmental conditions.

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.

Small RNA-based plant protection against diseases.

Plant diseases cause significant decreases in yield and quality of crops and consequently pose a very substantial threat to food security. In the continuous search for environmentally friendly crop protection, exploitation of RNA interferance machinery is showing promising results. It is well established that small RNAs (sRNAs) including microRNA (miRNA) and small interfering RNA (siRNA) are involved in the regulation of gene expression via both transcriptional and post-transcriptional RNA silencing. sRNAs from host plants can enter into pathogen cells during invasion and silence pathogen genes. This process has been exploited through Host-Induced Gene Silencing (HIGS), in which plant transgenes that produce sRNAs are engineered to silence pest and pathogen genes. Similarly, exogenously applied sRNAs can enter pest and pathogen cells, either directly or via the hosts, and silence target genes. This process has been exploited in Spray-Induced Gene Silencing (SIGS). Here, we focus on the role of sRNAs and review how they have recently been used against various plant pathogens through HIGS or SIGS-based methods and discuss advantages and drawbacks of these approaches.

The isothiocyanate sulforaphane induces respiratory burst oxidase homologue D-dependent reactive oxygen species production and regulates expression of stress response genes.

Sulforaphane (SFN) is an isothiocyanate-type phytomolecule present in crucifers, which is mainly synthesized in response to biotic stress. In animals, SFN incorporated in the diet has anticancer properties among others. The mechanism of action and signaling are well described in animals; however, little is known in plants. The goal in the present study is to elucidate components of the SFN signaling pathway, particularly the production of reactive oxygen species (ROS), and its effect on the transcriptome. Our results showed that in Arabidopsis, SFN causes ROS production exclusively through the action of the NADPH oxidase RBOH isoform D that requires calcium as a signaling component for the ROS production. To add to this, we also analyzed the effect of SFN on the transcriptome by RNAseq. We observed the highest expression increase for heat shock proteins (HSP) genes and also for genes associated with the response to oxidative stress. The upregulation of several genes linked to the biotic stress response confirms the interplay between SFN and this stress. In addition, SFN increases the levels of transcripts related to the response to abiotic stress, as well as phytohormones. Taken together, these results indicate that SFN induces an oxidative burst leading to signaling events. This oxidative burst may cause the increase of the expression of genes such as heat shock proteins to restore cellular homeostasis and genes that codify possible components of the signaling pathway and putative effectors.

Comparison of Antifungal Activity of Bacillus Strains against Fusarium graminearum In Vitro and In Planta.

Fusarium graminearum (Fg) causes Fusarium head blight (FHB) disease in wheat and barley. This pathogen produces mycotoxins including deoxynivalenol (DON), the T-2 and fumorisin B1. Translocation of the mycotoxins in grains causes important losses in yields and contributes to serious health problems in humans and livestock. We tested the Bacillus strains, two commercial, QST713 (Serenade®) and FZB24 (TAEGRO®) and one non-commercial strain EU07 as microbial biological control agents against the F. graminearum strain Fg-K1-4 both in vitro and in planta. The EU07 strain showed better performance in suppressing the growth of Fg-K1-4. Cell-free bacterial cultures displayed significant antagonistic activity on Fg-K1-4. Remarkably, heat and proteinase K treatment of bacterial broths did not reduce the antagonistic activity of Bacillus cultures. DON assays showed that Bacillus strain was not affected by the presence of DON in the media. Leaf and head infection assays using Brachypodium distachyon (Bd-21) indicated that EU07 inhibits Fg-K1-4 growth in vivo and promotes plant growth. Overall, the EU07 strain performed better, indicating that it could be explored for the molecular investigations and protection of cereal crops against FHB disease.

Hotspot siRNA Confers Plant Resistance against Viral Infection.

A hallmark of antiviral RNA interference (RNAi) is the production of viral small interfering RNA (vsiRNA). Profiling of vsiRNAs indicates that certain regions of viral RNA genome or transcribed viral RNA, dubbed vsiRNA hotspots, are more prone to RNAi-mediated cleavage for vsiRNA biogenesis. However, the biological relevance of hotspot vsiRNAs to the host innate defence against pathogens remains to be elucidated. Here, we show that direct targeting a hotspot by a synthetic vsiRNA confers host resistance to virus infection. Using Northern blotting and RNAseq, we obtained a profile of vsiRNAs of the African cassava mosaic virus (ACMV), a single-stranded DNA virus. Sense and anti-sense strands of small RNAs corresponding to a hotspot and a coldspot vsiRNA were synthesised. Co-inoculation of Nicotiana benthamiana with the double-stranded hotspot siRNA protected plants from ACMV infection, where viral DNA replication and accumulation of viral mRNA were undetectable. The sense or anti-sense strand of this hotspot vsiRNA, and the coldspot vsiRNA in both double-stranded and single-stranded formats possessed no activity in viral protection. We further demonstrated that the hotspot vsiRNA-mediated virus resistance had a threshold effect and required an active RDR6. These data show that hotspot vsiRNAs bear a functional significance on antiviral RNAi, suggesting that they may have the potential as an exogenous protection agent for controlling destructive viral diseases in plants.

Spinach-based RNA mimicking GFP in plant cells.

Spinach RNA-mimicking GFP (S-RMG) has been successfully used to monitor cellular RNAs including microRNAs in bacterium, yeast, and human cells. However, S-RMG has not been established in plants. In this study, we found that like bacterial, yeast, and human cellular tRNAs, plant tRNAs such as tRNALys can protect and/or stabilize the Spinach RNA aptamer interaction with the fluorophore DFHBI enabling detectable levels of green fluorescence to be emitted. The tRNALys-Spinach-tRNALys, once delivered into "chloroplast-free" onion epidermal cells can emit strong green fluorescence in the presence of DFHBI. Our results demonstrate for the first time that Spinach-based RNA visualization has the potential for in vivo monitoring of RNAs in plant cells.

Genomic-Assisted Marker Development Suitable for CsCvy-1 Selection in Cucumber Breeding.

Cucumber is a widely grown vegetable crop plant and a host to many different plant pathogens. Cucumber vein yellowing virus (CVYV) causes economic losses on cucumber crops in Mediterranean countries and in some part of India such as West Bengal and in African countries such as Sudan. CVYV is an RNA potyvirus transmitted mechanically and by whitefly (Bemisia tabaci) in a semipersistent manner. Control of this virus is heavily dependent on the management of the insect vector and breeding virus-resistant lines. DNA markers have been used widely in conventional plant breeding programs via marker-assisted selection (MAS). However, very few resistance sources against CVYV in cucumber exist, and also the lack of tightly linked molecular markers to these sources restricts the rapid generation of resistant lines. In this work, we used genomics coupled with the bulked segregant analysis method and generated the MAS-friendly Kompetitive allele specific PCR (KASP) markers suitable for CsCvy-1 selection in cucumber breeding using a segregating F2 mapping population and commercial plant lines. Variant analysis was performed to generate single-nucleotide polymorphism (SNP)-based markers for mapping the population and genotyping the commercial lines. We fine-mapped the region by generating new markers down to 101 kb with eight genes. We provided SNP data for this interval, which could be useful for breeding programs and cloning the candidate genes.

Molecular and functional characterization of the SBP-box transcription factor SPL-CNR in tomato fruit ripening and cell death.

SlSPL-CNR, an SBP-box transcription factor (TF) gene residing at the epimutant Colourless non-ripening (Cnr) locus, is involved in tomato ripening. This epimutant provides a unique model to investigate the (epi)genetic basis of fruit ripening. Here we report that SlSPL-CNR is a nucleus-localized protein with a distinct monopartite nuclear localization signal (NLS). It consists of four consecutive residues ' 30KRKR33' at the N-terminus of the protein. Mutation of the NLS abolishes SlSPL-CNR's ability to localize in the nucleus. SlSPL-CNR comprises two zinc-finger motifs (ZFMs) within the C-terminal SBP-box domain. Both ZFMs contribute to zinc-binding activity. SlSPL-CNR can induce cell death in tomato and tobacco, dependent on its nuclear localization. However, the two ZFMs have differential impacts on SlSPL-CNR's induction of severe necrosis or mild necrotic ringspot. NLS and ZFM mutants cannot complement Cnr fruits to ripen. SlSPL-CNR interacts with SlSnRK1. Virus-induced SlSnRK1 silencing leads to reduction in expression of ripening-related genes and inhibits ripening in tomato. We conclude that SlSPL-CNR is a multifunctional protein that consists of a distinct monopartite NLS, binds to zinc, and interacts with SlSnRK1 to affect cell death and tomato fruit ripening.

Foxtail mosaic virus-induced flowering assays in monocot crops.

Virus-induced flowering (VIF) exploits RNA or DNA viruses to express flowering time genes to induce flowering in plants. Such plant virus-based tools have recently attracted widespread attention for their fundamental and applied uses in flowering physiology and in accelerating breeding in dicotyledonous crops and woody fruit-trees. We now extend this technology to a monocot grass and a cereal crop. Using a Foxtail mosaic virus (FoMV)-based VIF system, dubbed FoMViF, we showed that expression of florigenic Flowering Locus T (FT) genes can promote early flowering and spikelet development in proso millet, a C4 grass species with potential as a nutritional food and biofuel resource, and in non-vernalized C3 wheat, a major food crop worldwide. Floral and spikelet/grain induction in the two monocot plants was caused by the virally expressed untagged or FLAG-tagged FT orthologs, and the florigenic activity of rice Hd3a was more pronounced than its dicotyledonous counterparts in proso millet. The FoMViF system is easy to use and its efficacy to induce flowering and early spikelet/grain production is high. In addition to proso millet and wheat, we envisage that FoMViF will be also applicable to many economically important monocotyledonous food and biofuel crops.

An RNAi suppressor activates in planta virus-mediated gene editing.

RNA-guided CRISPR/Cas9 technology has been developed for gene/genome editing (GE) in organisms across kingdoms. However, in planta delivery of the two core GE components, Cas9 and small guide RNA (sgRNA), often involves time-consuming and labor-intensive production of transgenic plants. Here we show that Foxtail mosaic virus, a monocot- and dicot-infecting potexvirus, can simultaneously express Cas9, sgRNA, and an RNAi suppressor to efficiently induce GE in Nicotiana benthamiana through a transgenic plant-free manner.

Small RNA inhibits infection by downy mildew pathogen Hyaloperonospora arabidopsidis.

Gene silencing exists in eukaryotic organisms as a conserved regulation of the gene expression mechanism. In general, small RNAs (sRNAs) are produced within the eukaryotic cells and incorporated into an RNA-induced silencing complex (RISC) within cells. However, exogenous sRNAs, once delivered into cells, can also silence target genes via the same RISC. Here, we explored this concept by targeting the Cellulose synthase A3 (CesA3) gene of Hyaloperonospora arabidopsidis (Hpa), the downy mildew pathogen of Arabidopsis thaliana. Hpa spore suspensions were mixed with sense or antisense sRNAs and inoculated onto susceptible Arabidopsis seedlings. While sense sRNAs had no obvious effect on Hpa pathogenicity, antisense sRNAs inhibited spore germination and hence infection. Such inhibition of infection was not race-specific, but dependent on the length and capping of sRNAs. Inhibition of infection by double stranded sRNA was more efficient than that observed with antisense sRNA. Thus, exogenous sRNA targeting conserved CesA3 could suppress Hpa infection in Arabidopsis, indicating the potential of this simple and efficient sRNA-based approach for deciphering gene functions in obligate biotrophic pathogens as well as for R-gene independent control of diseases in plants.

Mini review: Revisiting mobile RNA silencing in plants.

Non-cell autonomous RNA silencing can spread from cell to cell and over long-distances in animals and plants. This process is genetically determined and requires mobile RNA signals. Genetic requirement and molecular nature of the mobile signals for non-cell-autonomous RNA silencing were intensively investigated in past few decades. No consensus dogma for mobile silencing can be reached in plants, yet published data are sometimes inconsistent and controversial. Thus, the genetic requirements and molecular signals involved in plant mobile silencing are still poorly understood. This article revisits our present understanding of intercellular and systemic non-cell autonomous RNA silencing, and summarises current debates on RNA signals for mobile silencing. In particular, we discuss new evidence on siRNA mobility, a DCL2-dependent genetic network for mobile silencing and its potential biological relevance as well as 22 nt siRNA being a mobile signal for non-cell-autonomous silencing in both Arabidopsis and Nicotiana benthamiana. This sets up a new trend in unravelling genetic components and small RNA signal molecules for mobile silencing in (across) plants and other organisms of different kingdoms. Finally we raise several outstanding questions that need to be addressed in future plant silencing research.

Identifying molecular markers suitable for Frl selection in tomato breeding.

Modern plant breeding heavily relies on the use of molecular markers. In recent years, next generation sequencing (NGS) emerged as a powerful technology to discover DNA sequence polymorphisms and generate molecular markers very rapidly and cost effectively, accelerating the plant breeding programmes. A single dominant locus, Frl, in tomato provides resistance to the fungal pathogen Fusarium oxysporum f. sp. radicis-lycopersici (FORL), causative agent of Fusarium crown and root rot. In this study, we describe the generation of molecular markers associated with the Frl locus. An F2 mapping population between an FORL resistant and a susceptible cultivar was generated. NGS technology was then used to sequence the genomes of a susceptible and a resistant parent as well the genomes of bulked resistant and susceptible F2 lines. We zoomed into the Frl locus and mapped the locus to a 900 kb interval on chromosome 9. Polymorphic single-nucleotide polymorphisms (SNPs) within the interval were identified and markers co-segregating with the resistant phenotype were generated. Some of these markers were tested successfully with commercial tomato varieties indicating that they can be used for marker-assisted selection in large-scale breeding programmes.

A Suppressor/Avirulence Gene Combination in Hyaloperonospora arabidopsidis Determines Race Specificity in Arabidopsis thaliana.

The pathosystem of Arabidopsis thaliana and diploid biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa) has been a model for investigating the molecular basis of Flor's gene-for-gene hypothesis. The isolates Hpa-Noks1 and Hpa-Cala2 are virulent on Arabidopsis accession RMX-A02 whilst an F1 generated from a cross between these two isolates was avirulent. The F2 progeny segregated 3,1 (avirulent, virulent), indicating a single major effect AVR locus in this pathogen. SNP-based linkage mapping confirmed a single AVR locus within a 14 kb map interval containing two genes encoding putative effectors. The Hpa-Cala2 allele of one gene, designated H. arabidopsidiscryptic1 (HAC1), encodes a protein with a signal peptide and an RxLR/dEER motif, and triggers a defense response in RMX-A02. The second gene is heterozygous in Hpa-Cala2. One allele, designated Suppressor ofHAC1Cala2 (S-HAC1Cala2 ) encodes a protein with a signal peptide and a dKEE motif with no RxLR motif; the other allele (s-hac1Cala2 ) encodes a protein with a signal peptide, a dEEE motif and is divergent in sequence from the S-HAC1Cala2 allele. In selfed progeny from Hpa-Cala2, dominant S-HAC1Cala2 allele carrying progeny correlates with virulence in RMX-A02, whereas homozygous recessive s-hac1Cala2 carrying progeny were avirulent. Genetic investigations suggested other heterozygous suppressor loci might exist in the Hpa-Cala2 genome.

Comparative WGBS identifies genes that influence non-ripe phenotype in tomato epimutant Colourless non-ripening.

Whole-genome bisulfite sequencing (WGBS) allows single-base resolution and genome-wide profiling of DNA methylation in plants and animals. This technology provides a powerful tool to identify genes that are potentially controlled by dynamic changes of DNA methylation and demethylation. However, naturally occurring epimutants are rare and genes under epigenetic regulation as well as their biological relevances are often difficult to define. In tomato, fruit development and ripening are a complex process that involves epigenetic control. We have taken the advantage of the tomato epimutant Colourless non-ripening (Cnr) and performed comparative mining of the WGBS datasets for the Cnr and SlCMT3-silenced Cnr fruits. We compared DNA methylation profiles for the promoter sequences of approximately 5,000 bp immediately upstream of the coding region of a list of 20 genes. Differentially methylated regions were found for some of these genes. Virus-induced gene silencing (VIGS) of differentially methylated gene SlDET1 or SlPDS resulted in unusual brown pigmentation in Cnr fruits. These results suggest that comparative WGBS coupled with VIGS can be used to identify genes that may contribute to the colourless unripe phenotype of fruit in the Cnr epimutant.

Roles of Dicer-Like Proteins 2 and 4 in Intra- and Intercellular Antiviral Silencing.

RNA silencing is an innate antiviral mechanism conserved in organisms across kingdoms. Such a cellular defense involves DICER or DICER-LIKEs (DCLs) that process plant virus RNAs into viral small interfering RNAs (vsiRNAs). Plants encode four DCLs that play diverse roles in cell-autonomous intracellular virus-induced RNA silencing (known as VIGS) against viral invasion. VIGS can spread between cells. However, the genetic basis and involvement of vsiRNAs in non-cell-autonomous intercellular VIGS remains poorly understood. Using GFP as a reporter gene together with a suite of DCL RNAi transgenic lines, here we show that despite the well-established activities of DCLs in intracellular VIGS and vsiRNA biogenesis, DCL4 acts to inhibit intercellular VIGS whereas DCL2 is required (likely along with DCL2-processed/dependent vsiRNAs and their precursor RNAs) for efficient intercellular VIGS trafficking from epidermal to adjacent cells. DCL4 imposed an epistatic effect on DCL2 to impede cell-to-cell spread of VIGS. Our results reveal previously unknown functions for DCL2 and DCL4 that may form a dual defensive frontline for intra- and intercellular silencing to double-protect cells from virus infection in Nicotiana benthamiana.