Redox signaling and oxidative stress in systemic acquired resistance.

Plants fully depend on their immune systems to defend against pathogens. Upon pathogen attack, plants not only activate immune responses at the infection site but also trigger a defense mechanism known as systemic acquired resistance (SAR) in distal systemic tissues to prevent subsequent infections by a broad-spectrum of pathogens. SAR is induced by mobile signals produced at the infection site. Accumulating evidence suggests that reactive oxygen species (ROS) play a central role in SAR signaling. ROS burst at the infection site is one of the earliest cellular responses following pathogen infection and can spread to systemic tissues through membrane-associated NADPH oxidase-dependent relay-production of ROS. It is well known that ROS ignite redox signaling and when in excess, cause oxidative stress damaging cellular components. In this review, we summarize current knowledge on redox regulation of several SAR signaling components. We discuss the ROS amplification loop in systemic tissues involving multiple SAR mobile signals. Moreover, we highlight the essential role of oxidative stress in generating SAR signals including azelaic acid and extracellular NAD(P) [eNAD(P)]. Finally, we propose that eNAD(P) is a damage-associated molecular pattern serving as a converging point of SAR mobile signals in systemic tissues.

Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene.

Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both 'Carrizo' citrange and 'Duncan' grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.

Damage-Associated Molecular Patterns and Systemic Signaling.

Cellular damage inflicted by wounding, pathogen infection, and herbivory releases a variety of host-derived metabolites, degraded structural components, and peptides into the extracellular space that act as alarm signals when perceived by adjacent cells. These so-called damage-associated molecular patterns (DAMPs) function through plasma membrane localized pattern recognition receptors to regulate wound and immune responses. In plants, DAMPs act as elicitors themselves, often inducing immune outputs such as calcium influx, reactive oxygen species generation, defense gene expression, and phytohormone signaling. Consequently, DAMP perception results in a priming effect that enhances resistance against subsequent pathogen infections. Alongside their established function in local tissues, recent evidence supports a critical role of DAMP signaling in generation and/or amplification of mobile signals that induce systemic immune priming. Here, we summarize the identity, signaling, and synergy of proposed and established plant DAMPs, with a focus on those with published roles in systemic signaling.

N-hydroxypipecolic acid triggers systemic acquired resistance through extracellular NAD(P).

Systemic acquired resistance (SAR) is a long-lasting broad-spectrum plant defense mechanism induced in distal systemic tissues by mobile signals generated at the primary infection site. Despite the discoveries of multiple potential mobile signals, how these signals cooperate to trigger downstream SAR signaling is unknown. Here, we show that endogenous extracellular nicotinamide adenine dinucleotide (phosphate) [eNAD(P)] accumulates systemically upon pathogen infection and that both eNAD(P) and the lectin receptor kinase (LecRK), LecRK-VI.2, are required in systemic tissues for the establishment of SAR. Moreover, putative mobile signals, e.g., N-hydroxypipecolic acid (NHP), trigger de novo systemic eNAD(P) accumulation largely through the respiratory burst oxidase homolog RBOHF-produced reactive oxygen species (ROS). Importantly, NHP-induced systemic immunity mainly depends on ROS, eNAD(P), LecRK-VI.2, and BAK1, indicating that NHP induces SAR primarily through the ROS-eNAD(P)-LecRK-VI.2/BAK1 signaling pathway. Our results suggest that mobile signals converge on eNAD(P) in systemic tissues to trigger SAR through LecRK-VI.2.

Evaluating Nicotinamide Adenine Dinucleotide for Its Effects on Halo Blight of Snap Bean.

Halo blight, caused by Pseudomonas syringae pv. phaseolicola, is one of the major bacterial diseases on snap bean in Florida, and the outbreaks of this disease have occurred more often in recent years. Current management of this disease primarily depends on application of fixed copper-based bactericides but climate change and resistance development in the pathogen populations still cause hardship for management of this disease, especially in south Florida. In this study, nicotinamide adenine dinucleotide (NAD+) was evaluated in the greenhouse for its potential to reduce halo blight on snap bean. When NAD+ at 5 mM was applied by soil drench, foliar spray, or leaf infiltration, NAD+ significantly (P < 0.05) reduced disease severity of halo blight on snap bean compared with the untreated control. When NAD+ was applied by leaf infiltration, among the tested concentrations, NAD+ at 0.5 to 1.0 mM was most effective in decreasing halo blight disease. NAD+ at 2.5 mM applied as a foliar spray in rotation with Kocide 3000 (copper hydroxide) at 0.5 mg/ml further reduced disease severity compared with Kocide 3000 alone. In the in vitro study, no inhibitory effects of NAD+ were detected on the bacterial pathogen P. syringae pv. phaseolicola. Results of real-time PCR showed that the defense-related genes PR1, AZI1, EDS1, SARD1, PDF1.2, and PAL1 were upregulated in the NAD+ treatment. Taken together, these data indicated that NAD+ significantly suppressed halo blight on snap bean, and application of NAD+ has the potential in management of this important disease.

Efficient artificial microRNA vectors for gene silencing in citrus.

KEY MESSAGE: Three new artificial microRNA vectors were constructed and evaluated, and results showed that these vectors are highly efficient in the silencing of the citrus PHYTOENE DESATURASE gene.

Selection of transgenic citrus plants based on glyphosate tolerance conferred by a citrus 5-enolpyruvylshikimate-3-phosphate synthase variant.

KEY MESSAGE: We have defined the conditions for citrus transformations using glyphosate as selection agent. This protocol results in high transformation rate and low incidence of chimeric shoots. Glyphosate, the most widely used herbicide in the world, specifically inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an essential enzyme of the shikimate pathway. Various laboratory-generated or naturally evolved glyphosate-resistant EPSPS variants have been used to produce glyphosate-tolerant transgenic crops, enabling highly effective weed control in agriculture. In this study, we explored the potential of using a citrus EPSPS variant that mimics the previously reported Eleusine indica glyphosate-resistant TIPS (T102I + P106S) mutant for selection of transgenic citrus plants in the presence of glyphosate. We found that glyphosate did not suppress bud formation on 'Duncan' grapefruit seedling explants, but inhibited non-transgenic bud outgrowth to produce shoots in a concentration-dependent manner. At certain concentrations, glyphosate had dramatic effect on the transformation rate and the percentage of non-chimeric transgenic shoots in this newly developed selection system. Specifically, at 0, 10, 20, and 50 µM of glyphosate, the citrus TIPS EPSPS-based selection resulted in transformation rates of 4.02, 5.04, 14.46, and 40.78%, respectively, and 6.41, 23.96, 42.94, and 40.17% of non-chimeric transgenic shoots, respectively. These results indicate that the citrus TIPS EPSPS-glyphosate selection system is highly efficient and can be used as an alternative to antibiotic-based selection methods in citrus genetic transformation. Furthermore, the selection conditions defined in this study are expected to greatly facilitate the production of genetically modified, market-friendly citrus plants, such as cisgenic and intragenic plants.

Draft Genome Sequences of Pseudomonas syringae pv. tomato Strains J4 and J6, Isolated in Florida.

Pseudomonas syringae pv. tomato causes bacterial speck in tomato. We report the genome sequences of two P. syringae pv. tomato strains, J4 and J6, that are genetically closely related, with >99.9 average nucleotide identity (ANI), but vary in the presence of coronatine-associated genes.

Efficient CRISPR/Cas9 genome editing with Citrus embryogenic cell cultures.

BACKGROUND: Development of precise genome editing strategies is a prerequisite for producing edited plants that can aid in the study of gene function and help understand the genetic traits in a cultivar. Citrus embryogenic cell cultures can be used to rapidly produce a large population of genome edited transformed citrus lines. The ability to introduce specific mutations in the genome of these cells using two constructs (pC-PDS1 and pC-PDS2) was evaluated in this study. RESULTS: Citrus sinensis 'EV2' embryogenic cell cultures are amenable to Agrobacterium-mediated CRISPR/Cas9-based genome editing. Guide RNAs (gRNAs) targeting two locations in the phytoene desaturase (PDS) gene were either driven by the Arabidopsis U6-26 promoter (pC-PDS1) or assembled as a Csy4 array under the control of the CmYLCV promoter (pC-PDS2). All transgenic embryos were completely albino and no variegated phenotype was observed. We evaluated 12 lines from each construct in this study and the majority contain either insertion (1-2 bp), substitution (1 bp), or deletion (1-3 bp) mutations that occurred close to the protospacer adjacent motif. CONCLUSIONS: Both the pC-PDS1 and pC-PDS2 could successfully edit the citrus embryogenic cell cultures. However, the editing efficiency was dependent on the gRNA, confirming that the selection of a proper gRNA is essential for successful genome editing using the CRISPR/Cas9 technique. Also, utilization of embryogenic cell cultures offers another option for successful genome editing in citrus.

Differential Quantitative Requirements for NPR1 Between Basal Immunity and Systemic Acquired Resistance in Arabidopsis thaliana.

Non-expressor of pathogenesis-related (PR) genes1 (NPR1) is a key transcription coactivator of plant basal immunity and systemic acquired resistance (SAR). Two mutant alleles, npr1-1 and npr1-3, have been extensively used for dissecting the role of NPR1 in various signaling pathways. However, it is unknown whether npr1-1 and npr1-3 are null mutants. Moreover, the NPR1 transcript levels are induced two- to threefold upon pathogen infection or salicylic acid (SA) treatment, but the biological relevance of the induction is unclear. Here, we used molecular and biochemical approaches including quantitative PCR, immunoblot analysis, site-directed mutagenesis, and CRISPR/Cas9-mediated gene editing to address these questions. We show that npr1-3 is a potential null mutant, whereas npr1-1 is not. We also demonstrated that a truncated npr1 protein longer than the hypothesized npr1-3 protein is not active in SA signaling. Furthermore, we revealed that TGACG-binding (TGA) factors are required for NPR1 induction, but the reverse TGA box in the 5'UTR of NPR1 is dispensable for the induction. Finally, we show that full induction of NPR1 is required for basal immunity, but not for SAR, whereas sufficient basal transcription is essential for full-scale establishment of SAR. Our results indicate that induced transcript accumulation may be differentially required for different functions of a specific gene. Moreover, as npr1-1 is not a null mutant, we recommend that future research should use npr1-3 and potential null T-DNA insertion mutants for dissecting NPR1's function in various physiopathological processes.

Development of Improved Fruit, Vegetable, and Ornamental Crops Using the CRISPR/Cas9 Genome Editing Technique.

Horticultural crops, including fruit, vegetable, and ornamental plants are an important component of the agriculture production systems and play an important role in sustaining human life. With a steady growth in the world's population and the consequent need for more food, sustainable and increased fruit and vegetable crop production is a major challenge to guarantee future food security. Although conventional breeding techniques have significantly contributed to the development of important varieties, new approaches are required to further improve horticultural crop production. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has emerged as a valuable genome-editing tool able to change DNA sequences at precisely chosen loci. The CRISPR/Cas9 system was developed based on the bacterial adaptive immune system and comprises of an endonuclease guided by one or more single-guide RNAs to generate double-strand breaks. These breaks can then be repaired by the natural cellular repair mechanisms, during which genetic mutations are introduced. In a short time, the CRISPR/Cas9 system has become a popular genome-editing technique, with numerous examples of gene mutation and transcriptional regulation control in both model and crop plants. In this review, various aspects of the CRISPR/Cas9 system are explored, including a general presentation of the function of the CRISPR/Cas9 system in bacteria and its practical application as a biotechnological tool for editing plant genomes, particularly in horticultural crops.

Extracellular pyridine nucleotides trigger plant systemic immunity through a lectin receptor kinase/BAK1 complex.

Systemic acquired resistance (SAR) is a long-lasting broad-spectrum plant immunity induced by mobile signals produced in the local leaves where the initial infection occurs. Although multiple structurally unrelated signals have been proposed, the mechanisms responsible for perception of these signals in the systemic leaves are unknown. Here, we show that exogenously applied nicotinamide adenine dinucleotide (NAD+) moves systemically and induces systemic immunity. We demonstrate that the lectin receptor kinase (LecRK), LecRK-VI.2, is a potential receptor for extracellular NAD+ (eNAD+) and NAD+ phosphate (eNADP+) and plays a central role in biological induction of SAR. LecRK-VI.2 constitutively associates with BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) in vivo. Furthermore, BAK1 and its homolog BAK1-LIKE1 are required for eNAD(P)+ signaling and SAR, and the kinase activities of LecR-VI.2 and BAK1 are indispensable to their function in SAR. Our results indicate that eNAD+ is a putative mobile signal, which triggers SAR through its receptor complex LecRK-VI.2/BAK1 in Arabidopsis thaliana.

Exogenous Nicotinamide Adenine Dinucleotide Induces Resistance to Citrus Canker in Citrus.

Nicotinamide adenine dinucleotide (NAD) is a universal electron carrier that participates in important intracellular metabolic reactions and signaling events. Interestingly, emerging evidence in animals indicates that cellular NAD can be actively or passively released into the extracellular space, where it is processed or perceived by ectoenzymes or cell-surface receptors. We have recently shown in Arabidopsis thaliana that exogenous NAD induces defense responses, that pathogen infection leads to release of NAD into the extracellular space at concentrations sufficient for defense activation, and that depletion of extracellular NAD (eNAD) by transgenic expression of the human NAD-hydrolyzing ectoenzyme CD38 inhibits plant immunity. We therefore hypothesize that, during plant-microbe interactions, NAD is released from dead or dying cells into the extracellular space where it interacts with adjacent naïve cells' surface receptors, which in turn activate downstream immune signaling. However, it is currently unknown whether eNAD signaling is unique to Arabidopsis or the Brassicaceae family. In this study, we treated citrus plants with exogenous NAD+ and tested NAD+-induced transcriptional changes and disease resistance. Our results show that NAD+ induces profound transcriptome changes and strong resistance to citrus canker, a serious citrus disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc). Furthermore, NAD+-induced resistance persists in new flushes emerging after removal of the tissues previously treated with NAD+. Finally, NAD+ treatment primes citrus tissues, resulting in a faster and stronger induction of multiple salicylic acid pathway genes upon subsequent Xcc infection. Taken together, these results indicate that exogenous NAD+ is able to induce immune responses in citrus and suggest that eNAD may also be an elicitor in this woody plant species.

The Arabidopsis Elongator Subunit ELP3 and ELP4 Confer Resistance to Bacterial Speck in Tomato.

Although production of tomato (Solanum lycopersicum) is threatened by a number of major diseases worldwide, it has been difficult to identify effective and durable management measures against these diseases. In this study, we attempted to improve tomato disease resistance by transgenic overexpression of genes encoding the Arabidopsis thaliana Elongator (AtELP) complex subunits AtELP3 and AtELP4. We show that overexpression of AtELP3 and AtELP4 significantly enhanced resistance to tomato bacterial speck caused by the Pseudomonas syringae pv. tomato strain J4 (Pst J4) without clear detrimental effects on plant growth and development. Interestingly, the transgenic plants exhibited resistance to Pst J4 only when inoculated through foliar sprays but not through infiltration into the leaf apoplast. Although this result suggested possible involvement of stomatal immunity, we found that Pst J4 inoculation did not induce stomatal closure and there were no differences in stomatal apertures and conductance between the transgenic and control plants. Further RNA sequencing and real-time quantitative PCR analyses revealed a group of defense-related genes to be induced to higher levels after infection in the AtELP4 transgenic tomato plants than in the control, suggesting that the enhanced disease resistance of the transgenic plants may be attributed to elevated induction of defense responses. Additionally, we show that the tomato genome contains single-copy genes encoding all six Elongator subunits (SlELPs), which share high identities with the AtELP proteins, and that SlELP3 and SlELP4 complemented the Arabidopsis Atelp3 and Atelp4 mutants, respectively, indicating that the function of tomato Elongator is probably conserved. Taken together, our results not only shed new light on the tomato Elongator complex, but also revealed potential candidate genes for engineering disease resistance in tomato.

NPR1 as a transgenic crop protection strategy in horticultural species.

The NPR1 (NONEXPRESSOR OF PATHOGENESIS RELATED GENES1) gene has a central role in the long-lasting, broad-spectrum defense response known as systemic acquired resistance (SAR). When overexpressed in a transgenic context in Arabidopsis thaliana, this gene enhances resistance to a number of biotic and abiotic stresses. Its position as a key regulator of defense across diverse plant species makes NPR1 a strong candidate gene for genetic engineering disease and stress tolerance into other crops. High-value horticultural crops face many new challenges from pests and pathogens, and their emergence exceeds the pace of traditional breeding, making the application of NPR1-based strategies potentially useful in fruit and vegetable crops. However, plants overexpressing NPR1 occasionally present detrimental morphological traits that make its application less attractive. The practical utility of NPR-based approaches will be a balance of resistance gains versus other losses. In this review, we summarize the progress on the understanding of NPR1-centered applications in horticultural and other crop plants. We also discuss the effect of the ectopic expression of the A. thaliana NPR1 gene and its orthologs in crop plants and outline the future challenges of using NPR1 in agricultural applications.

The Elongator complex-associated protein DRL1 plays a positive role in immune responses against necrotrophic fungal pathogens in Arabidopsis.

DEFORMED ROOT AND LEAVES1 (DRL1) is an Arabidopsis homologue of the yeast TOXIN TARGET4 (TOT4)/KILLER TOXIN-INSENSITIVE12 (KTI12) protein that is physically associated with the RNA polymerase II-interacting protein complex named Elongator. Mutations in DRL1 and Elongator lead to similar morphological and molecular phenotypes, suggesting that DRL1 and Elongator may functionally overlap in Arabidopsis. We have shown previously that Elongator plays an important role in both salicylic acid (SA)- and jasmonic acid (JA)/ethylene (ET)-mediated defence responses. Here, we tested whether DRL1 also plays a similar role as Elongator in plant immune responses. Our results show that, although DRL1 partially contributes to SA-induced cytotoxicity, it does not play a significant role in SA-mediated expression of PATHOGENESIS-RELATED genes and resistance to the virulent bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. In contrast, DRL1 is required for JA/ET- and necrotrophic fungal pathogen Botrytis cinerea-induced defence gene expression and for resistance to B. cinerea and Alternaria brassicicola. Furthermore, unlike the TOT4/KTI12 gene which, when overexpressed in yeast, confers zymocin resistance, a phenotype of the tot4/kti12 mutant, overexpression of DRL1 does not change B. cinerea-induced defence gene expression and resistance to this pathogen. Finally, DRL1 contains an N-terminal P-loop and a C-terminal calmodulin (CaM)-binding domain and is a CaM-binding protein. We demonstrate that both the P-loop and the CaM-binding domain are essential for the function of DRL1 in B. cinerea-induced expression of PDF1.2 and ORA59, and in resistance to B. cinerea, suggesting that the function of DRL1 in plant immunity may be regulated by ATP/GTP and CaM binding.