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.

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.

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.

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.

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.

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.

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.

Timing of plant immune responses by a central circadian regulator.

The principal immune mechanism against biotrophic pathogens in plants is the resistance (R)-gene-mediated defence. It was proposed to share components with the broad-spectrum basal defence machinery. However, the underlying molecular mechanism is largely unknown. Here we report the identification of novel genes involved in R-gene-mediated resistance against downy mildew in Arabidopsis and their regulatory control by the circadian regulator, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1). Numerical clustering based on phenotypes of these gene mutants revealed that programmed cell death (PCD) is the major contributor to resistance. Mutants compromised in the R-gene-mediated PCD were also defective in basal resistance, establishing an interconnection between these two distinct defence mechanisms. Surprisingly, we found that these new defence genes are under circadian control by CCA1, allowing plants to 'anticipate' infection at dawn when the pathogen normally disperses the spores and time immune responses according to the perception of different pathogenic signals upon infection. Temporal control of the defence genes by CCA1 differentiates their involvement in basal and R-gene-mediated defence. Our study has revealed a key functional link between the circadian clock and plant immunity.

Involvement of the electrophilic isothiocyanate sulforaphane in Arabidopsis local defense responses.

Plants defend themselves against microbial pathogens through a range of highly sophisticated and integrated molecular systems. Recognition of pathogen-secreted effector proteins often triggers the hypersensitive response (HR), a complex multicellular defense reaction where programmed cell death of cells surrounding the primary site of infection is a prominent feature. Even though the HR was described almost a century ago, cell-to-cell factors acting at the local level generating the full defense reaction have remained obscure. In this study, we sought to identify diffusible molecules produced during the HR that could induce cell death in naive tissue. We found that 4-methylsulfinylbutyl isothiocyanate (sulforaphane) is released by Arabidopsis (Arabidopsis thaliana) leaf tissue undergoing the HR and that this compound induces cell death as well as primes defense in naive tissue. Two different mutants impaired in the pathogen-induced accumulation of sulforaphane displayed attenuated programmed cell death upon bacterial and oomycete effector recognition as well as decreased resistance to several isolates of the plant pathogen Hyaloperonospora arabidopsidis. Treatment with sulforaphane provided protection against a virulent H. arabidopsidis isolate. Glucosinolate breakdown products are recognized as antifeeding compounds toward insects and recently also as intracellular signaling and bacteriostatic molecules in Arabidopsis. The data presented here indicate that these compounds also trigger local defense responses in Arabidopsis tissue.

Arabidopsis receptor-like protein30 and receptor-like kinase suppressor of BIR1-1/EVERSHED mediate innate immunity to necrotrophic fungi.

Effective plant defense strategies rely in part on the perception of non-self determinants, so-called microbe-associated molecular patterns (MAMPs), by transmembrane pattern recognition receptors leading to MAMP-triggered immunity. Plant resistance against necrotrophic pathogens with a broad host range is complex and yet not well understood. Particularly, it is unclear if resistance to necrotrophs involves pattern recognition receptors. Here, we partially purified a novel proteinaceous elicitor called sclerotinia culture filtrate elicitor1 (SCFE1) from the necrotrophic fungal pathogen Sclerotinia sclerotiorum that induces typical MAMP-triggered immune responses in Arabidopsis thaliana. Analysis of natural genetic variation revealed five Arabidopsis accessions (Mt-0, Lov-1, Lov-5, Br-0, and Sq-1) that are fully insensitive to the SCFE1-containing fraction. We used a forward genetics approach and mapped the locus determining SCFE1 sensitivity to receptor-like protein30 (RLP30). We also show that SCFE1-triggered immune responses engage a signaling pathway dependent on the regulatory receptor-like kinases brassinosteroid insensitive1-associated receptor kinase1 (BAK1) and Suppressor of BIR1-1/evershed (SOBIR1/EVR). Mutants of RLP30, BAK1, and SOBIR1 are more susceptible to S. sclerotiorum and the related fungus Botrytis cinerea. The presence of an elicitor in S. sclerotiorum evoking MAMP-triggered immune responses and sensed by RLP30/SOBIR1/BAK1 demonstrates the relevance of MAMP-triggered immunity in resistance to necrotrophic fungi.

Role of the penetration-resistance genes PEN1, PEN2 and PEN3 in the hypersensitive response and race-specific resistance in Arabidopsis thaliana.

Plants are highly capable of recognizing and defending themselves against invading microbes. Adapted plant pathogens secrete effector molecules to suppress the host's immune system. These molecules may be recognized by host-encoded resistance proteins, which then trigger defense in the form of the hypersensitive response (HR) leading to programmed cell death of the host tissue at the infection site. The three proteins PEN1, PEN2 and PEN3 have been found to act as central components in cell wall-based defense against the non-adapted powdery mildew Blumeria graminis fsp. hordei (Bgh). We found that loss of function mutations in any of the three PEN genes cause decreased hypersensitive cell death triggered by recognition of effectors from oomycete and bacterial pathogens in Arabidopsis. There were considerable additive effects of the mutations. The HR induced by recognition of AvrRpm1 was almost completely abolished in the pen2 pen3 and pen1 pen3 double mutants and the loss of cell death could be linked to indole glucosinolate breakdown products. However, the loss of the HR in pen double mutants did not affect the plants' ability to restrict bacterial growth, whereas resistance to avirulent isolates of the oomycete Hyaloperonospora arabidopsidis was strongly compromised. In contrast, the double and triple mutants demonstrated varying degrees of run-away cell death in response to Bgh. Taken together, our results indicate that the three genes PEN1, PEN2 and PEN3 extend in functionality beyond their previously recognized functions in cell wall-based defense against non-host pathogens.

A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening.

A major component in the regulatory network controlling fruit ripening is likely to be the gene at the tomato Colorless non-ripening (Cnr) locus. The Cnr mutation results in colorless fruits with a substantial loss of cell-to-cell adhesion. The nature of the mutation and the identity of the Cnr gene were previously unknown. Using positional cloning and virus-induced gene silencing, here we demonstrate that an SBP-box (SQUAMOSA promoter binding protein-like) gene resides at the Cnr locus. Furthermore, the Cnr phenotype results from a spontaneous epigenetic change in the SBP-box promoter. The discovery that Cnr is an epimutation was unexpected, as very few spontaneous epimutations have been described in plants. This study demonstrates that an SBP-box gene is critical for normal ripening and highlights the likely importance of epialleles in plant development and the generation of natural variation.

The Arabidopsis leucine-rich repeat receptor-like kinases BAK1/SERK3 and BKK1/SERK4 are required for innate immunity to hemibiotrophic and biotrophic pathogens.

Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) constitutes an important layer of innate immunity in plants. The leucine-rich repeat (LRR) receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN SENSING2 (FLS2) are the PRRs for the peptide PAMPs elf18 and flg22, which are derived from bacterial EF-Tu and flagellin, respectively. Using coimmunoprecipitation and mass spectrometry analyses, we demonstrated that EFR and FLS2 undergo ligand-induced heteromerization in planta with several LRR receptor-like kinases that belong to the SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, including BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1/SERK3 (BAK1/SERK3) and BAK1-LIKE1/SERK4 (BKK1/SERK4). Using a novel bak1 allele that does not exhibit pleiotropic defects in brassinosteroid and cell death responses, we determined that BAK1 and BKK1 cooperate genetically to achieve full signaling capability in response to elf18 and flg22 and to the damage-associated molecular pattern AtPep1. Furthermore, we demonstrated that BAK1 and BKK1 contribute to disease resistance against the hemibiotrophic bacterium Pseudomonas syringae and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis. Our work reveals that the establishment of PAMP-triggered immunity (PTI) relies on the rapid ligand-induced recruitment of multiple SERKs within PRR complexes and provides insight into the early PTI signaling events underlying this important layer of plant innate immunity.

Genome sequence of the plant-growth-promoting bacterium Bacillus velezensis EU07.

Many Gram-positive spore-forming rhizobacteria of the genus Bacillus show potential as biocontrol biopesticides that promise improved sustainability and ecological safety in agriculture. Here, we present a draft-quality genome sequence for Bacillus velezensis EU07, which shows growth-promotion in tomato plants and biocontrol against Fusarium head blight. We found that the genome of EU07 is almost identical to that of the commercially used strain QST713, but identified 46 single-nucleotide differences that distinguish these strains from each other. The availability of this genome sequence will facilitate future efforts to unravel the genetic and molecular basis for EU07's beneficial properties.

Antiviral defense in plant stem cells.

Undifferentiated plant and animal stem cells are essential for cell, tissue, and organ differentiation, development, and growth. They possess unusual antiviral immunity which differs from that in specialized cells. By comparison to animal stem cells, we discuss how plant stem cells defend against viral invasion and beyond.

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.

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.

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.