Extracellular ATP Acts on Jasmonate Signaling to Reinforce Plant Defense.

Damaged cells send various signals to stimulate defense responses. Recent identification and genetic studies of the plant purinoceptor, P2K1 (also known as DORN1), have demonstrated that extracellular ATP is a signal involved in plant stress responses, including wounding, perhaps to evoke plant defense. However, it remains largely unknown how extracellular ATP induces plant defense responses. Here, we demonstrate that extracellular ATP induces plant defense mediated through activation of the intracellular signaling of jasmonate (JA), a well-characterized defense hormone. In Arabidopsis (Arabidopsis thaliana) leaves, ATP pretreatment induced resistance against the necrotrophic fungus, Botrytis cinerea The induced resistance was enhanced in the P2K1 receptor overexpression line, but reduced in the receptor mutant, dorn1-3 Mining the transcriptome data revealed that ATP induces a set of JA-induced genes. In addition, the P2K1-associated coexpression network contains defense-related genes, including those encoding jasmonate ZIM-domain (JAZ) proteins, which play key roles as repressors of JA signaling. We examined whether extracellular ATP impacts the stability of JAZ1 in Arabidopsis. The results showed that the JAZ1 stability decreased in response to ATP addition in a proteasome-dependent manner. This reduction required intracellular signaling via second messengers-cytosolic calcium, reactive oxygen species, and nitric oxide. Interestingly, the ATP-induced JAZ1 degradation was attenuated in the JA receptor mutant, coi1, but not in the JA biosynthesis mutant, aos, or upon addition of JA biosynthesis inhibitors. Immunoprecipitation analysis demonstrated that ATP increases the interaction between COI1 and JAZ1, suggesting direct cross talk between extracellular ATP and JA in intracellular signaling events. Taken together, these results suggest that extracellular ATP signaling directly impacts the JA signaling pathway to maximize plant defense responses.

Oxidative and Glycation Damage to Mitochondrial DNA and Plastid DNA during Plant Development.

Oxidative damage to plant proteins, lipids, and DNA caused by reactive oxygen species (ROS) has long been studied. The damaging effects of reactive carbonyl groups (glycation damage) to plant proteins and lipids have also been extensively studied, but only recently has glycation damage to the DNA in plant mitochondria and plastids been reported. Here, we review data on organellar DNA maintenance after damage from ROS and glycation. Our focus is maize, where tissues representing the entire range of leaf development are readily obtained, from slow-growing cells in the basal meristem, containing immature organelles with pristine DNA, to fast-growing leaf cells, containing mature organelles with highly-fragmented DNA. The relative contributions to DNA damage from oxidation and glycation are not known. However, the changing patterns of damage and damage-defense during leaf development indicate tight coordination of responses to oxidation and glycation events. Future efforts should be directed at the mechanism by which this coordination is achieved.

Ribonucleotide and R-Loop Damage in Plastid DNA and Mitochondrial DNA during Maize Development.

Although the temporary presence of ribonucleotides in DNA is normal, their persistence represents a form of DNA damage. Here, we assess such damage and damage defense to DNA in plastids and mitochondria of maize. Shoot development proceeds from meristematic, non-pigmented cells containing proplastids and promitochondria at the leaf base to non-dividing green cells in the leaf blade containing mature organelles. The organellar DNAs (orgDNAs) become fragmented during this transition. Previously, orgDNA damage and damage defense of two types, oxidative and glycation, was described in maize, and now a third type, ribonucleotide damage, is reported. We hypothesized that ribonucleotide damage changes during leaf development and could contribute to the demise of orgDNAs. The levels of ribonucleotides and R-loops in orgDNAs and of RNase H proteins in organelles were measured throughout leaf development and in leaves grown in light and dark conditions. The data reveal that ribonucleotide damage to orgDNAs increased by about 2- to 5-fold during normal maize development from basal meristem to green leaf and when leaves were grown in normal light conditions compared to in the dark. During this developmental transition, the levels of the major agent of defense, RNase H, declined. The decline in organellar genome integrity during maize development may be attributed to oxidative, glycation, and ribonucleotide damages that are not repaired.

The intrinsically disordered C-terminus of purinoceptor P2K1 fine-tunes plant responses to extracellular ATP.

P2K1 is a plant-specific purinoceptor that perceives extracellular ATP (eATP), a signaling molecular implicated in various physiological processes. Interestingly, P2K1 harbors a C-terminal intrinsically disordered region (IDR). When we overexpressed a truncated P2K1 (P2K1t ) lacking the IDR, primary root growth completely ceased in response to eATP. We investigated the functional roles of the IDR in P2K1 using a combination of molecular genetics, calcium imaging, gene expression analysis, and histochemical approaches. We found that the P2K1t variant gave rise to an amplified response to eATP, through accumulation of superoxide, altered cell wall integrity, and ultimate cell death in the primary root tip. Together, these observations underscore the significant involvement of the C-terminal tail of P2K1 in root growth regulation.

Analysis of the Plastid Genome Sequence During Maize Seedling Development.

Shoot development in maize progresses from small, non-pigmented meristematic cells to expanded cells in the green leaf. During this transition, large plastid DNA (ptDNA) molecules in proplastids become fragmented in the photosynthetically-active chloroplasts. The genome sequences were determined for ptDNA obtained from Zea mays B73 plastids isolated from four tissues: base of the stalk (the meristem region); fully-developed first green leaf; first three leaves from light-grown seedlings; and first three leaves from dark-grown (etiolated) seedlings. These genome sequences were then compared to the Z. mays B73 plastid reference genome sequence that was previously obtained from green leaves. The assembled plastid genome was identical among these four tissues to the reference genome. Furthermore, there was no difference among these tissues in the sequence at and around the previously documented 27 RNA editing sites. There were, however, more sequence variants (insertions/deletions and single-nucleotide polymorphisms) for leaves grown in the dark than in the light. These variants were tightly clustered into two areas within the inverted repeat regions of the plastid genome. We propose a model for how these variant clusters could be generated by replication-transcription conflict.

Glycation damage to organelles and their DNA increases during maize seedling development.

Shoot development in maize begins when meristematic, non-pigmented cells at leaf base stop dividing and proceeds toward the expanded green cells of the leaf blade. During this transition, promitochondria and proplastids develop into mature organelles and their DNA becomes fragmented. Changes in glycation damage during organelle development were measured for protein and DNA, as well as the glycating agent methyl glyoxal and the glycation-defense protein DJ-1 (known as Park7 in humans). Maize seedlings were grown under normal, non-stressful conditions. Nonetheless, we found that glycation damage, as well as defenses against glycation, follow the same developmental pattern we found previously for reactive oxygen species (ROS): as damage increases, damage-defense measures decrease. In addition, light-grown leaves had more glycation and less DJ-1 compared to dark-grown leaves. The demise of maize organellar DNA during development may therefore be attributed to both oxidative and glycation damage that is not repaired. The coordination between oxidative and glycation damage, as well as damage-response from the nucleus is also discussed.

Purinoceptor P2K1/DORN1 Enhances Plant Resistance Against a Soilborne Fungal Pathogen, Rhizoctonia solani.

The purinoceptor P2K1/DORN1 recognizes extracellular ATP, a damage-associated molecular pattern (DAMP) released upon cellular disruption by wounding and necrosis, which in turn, boost plant innate immunity. P2K1 is known to confer plant resistance to foliar biotrophic, hemi-biotrophic, and necrotrophic pathogens. However, until now, no information was available on its function in defense against root pathogens. In this report, we describe the contribution of P2K1 to resistance in Arabidopsis against Rhizoctonia solani, a broad host range, necrotrophic soilborne fungal pathogen. In pot assays, the Arabidopsis P2K1 overexpression line OxP2K1 showed longer root length and a greater rosette surface area than wild type in the presence of the pathogen. In contrast, the knockout mutant dorn1-3 and the double mutant rbohd/f, defective in two subunits of the respiratory burst complex NADPH oxidase, exhibited significant reductions in shoot and root lengths and rosette surface area compared to wild type when the pathogen was present. Expression of PR1, PDF1.2, and JAZ5 in the roots was reduced in dorn1-3 and rbohd/f and elevated in OxP2K1 relative to wild type, indicating that the salicylate and jasmonate defense signaling pathways functioned in resistance. These results indicated that a DAMP-mediated defense system confers basal resistance against an important root necrotrophic fungal pathogen.

Reactive Oxygen Species, Antioxidant Agents, and DNA Damage in Developing Maize Mitochondria and Plastids.

Maize shoot development progresses from non-pigmented meristematic cells at the base of the leaf to expanded and non-dividing green cells of the leaf blade. This transition is accompanied by the conversion of promitochondria and proplastids to their mature forms and massive fragmentation of both mitochondrial DNA (mtDNA) and plastid DNA (ptDNA), collectively termed organellar DNA (orgDNA). We measured developmental changes in reactive oxygen species (ROS), which at high concentrations can lead to oxidative stress and DNA damage, as well as antioxidant agents and oxidative damage in orgDNA. Our plants were grown under normal, non-stressful conditions. Nonetheless, we found more oxidative damage in orgDNA from leaf than stalk tissues and higher levels of hydrogen peroxide, superoxide, and superoxide dismutase in leaf than stalk tissues and in light-grown compared to dark-grown leaves. In both mitochondria and plastids, activities of the antioxidant enzyme peroxidase were higher in stalk than in leaves and in dark-grown than light-grown leaves. In protoplasts, the amount of the small-molecule antioxidants, glutathione and ascorbic acid, and catalase activity were also higher in the stalk than in leaf tissue. The data suggest that the degree of oxidative stress in the organelles is lower in stalk than leaf and lower in dark than light growth conditions. We speculate that the damaged/fragmented orgDNA in leaves (but not the basal meristem) results from ROS signaling to the nucleus to stop delivering DNA repair proteins to mature organelles producing large amounts of ROS.

ßC1, pathogenicity determinant encoded by Cotton leaf curl Multan betasatellite, interacts with calmodulin-like protein 11 (Gh-CML11) in Gossypium hirsutum.

Begomoviruses interfere with host plant machinery to evade host defense mechanism by interacting with plant proteins. In the old world, this group of viruses are usually associated with betasatellite that induces severe disease symptoms by encoding a protein, ßC1, which is a pathogenicity determinant. Here, we show that ßC1 encoded by Cotton leaf curl Multan betasatellite (CLCuMB) requires Gossypium hirsutum calmodulin-like protein 11 (Gh-CML11) to infect cotton. First, we used the in silico approach to predict the interaction of CLCuMB-ßC1 with Gh-CML11. A number of sequence- and structure-based in-silico interaction prediction techniques suggested a strong putative binding of CLCuMB-ßC1 with Gh-CML11 in a Ca+2-dependent manner. In-silico interaction prediction was then confirmed by three different experimental approaches: The Gh-CML11 interaction was confirmed using CLCuMB-ßC1 in a yeast two hybrid system and pull down assay. These results were further validated using bimolecular fluorescence complementation system showing the interaction in cytoplasmic veins of Nicotiana benthamiana. Bioinformatics and molecular studies suggested that CLCuMB-ßC1 induces the overexpression of Gh-CML11 protein and ultimately provides calcium as a nutrient source for virus movement and transmission. This is the first comprehensive study on the interaction between CLCuMB-ßC1 and Gh-CML11 proteins which provided insights into our understating of the role of ßC1 in cotton leaf curl disease.

In silico Prediction and Validations of Domains Involved in Gossypium hirsutum SnRK1 Protein Interaction With Cotton Leaf Curl Multan Betasatellite Encoded ßC1.

Cotton leaf curl disease (CLCuD) caused by viruses of genus Begomovirus is a major constraint to cotton (Gossypium hirsutum) production in many cotton-growing regions of the world. Symptoms of the disease are caused by Cotton leaf curl Multan betasatellite (CLCuMB) that encodes a pathogenicity determinant protein, ßC1. Here, we report the identification of interacting regions in ßC1 protein by using computational approaches including sequence recognition, and binding site and interface prediction methods. We show the domain-level interactions based on the structural analysis of G. hirsutum SnRK1 protein and its domains with CLCuMB-ßC1. To verify and validate the in silico predictions, three different experimental approaches, yeast two hybrid, bimolecular fluorescence complementation and pull down assay were used. Our results showed that ubiquitin-associated domain (UBA) and autoinhibitory sequence (AIS) domains of G. hirsutum-encoded SnRK1 are involved in CLCuMB-ßC1 interaction. This is the first comprehensive investigation that combined in silico interaction prediction followed by experimental validation of interaction between CLCuMB-ßC1 and a host protein. We demonstrated that data from computational biology could provide binding site information between CLCuD-associated viruses/satellites and new hosts that lack known binding site information for protein-protein interaction studies. Implications of these findings are discussed.

A crosstalk between extracellular ATP and jasmonate signaling pathways for plant defense.

Damage-associated molecular patterns (DAMPs), such as extracellular ATP, act as danger signals in response to biotic and abiotic stresses. Extracellular ATP is perceived by a plant purinoceptor, P2 receptor kinase 1 (P2K1), inducing downstream signaling for defense responses. How ATP induces these defense responses has not been well studied. A recent study by Tripathi et al. (Plant Physiology, 176: 511-523, 2018) revealed a synergistic interaction between extracellular ATP and jasmonate (JA) signaling during plant defense responses. This signaling crosstalk requires the formation of secondary messengers, i.e., cytosolic calcium, reactive oxygen species, and nitric oxide. This finding has given a new direction towards understanding the defense signals activated by DAMPs. In this addendum, we discuss possible insights into how extracellular ATP signaling interacts with the JA signaling pathway for plant defense responses.

Extracellular Alkalinization as a Defense Response in Potato Cells.

A quantitative and robust bioassay to assess plant defense response is important for studies of disease resistance and also for the early identification of disease during pre- or non-symptomatic phases. An increase in extracellular pH is known to be an early defense response in plants. In this study, we demonstrate extracellular alkalinization as a defense response in potatoes. Using potato suspension cell cultures, we observed an alkalinization response against various pathogen- and plant-derived elicitors in a dose- and time-dependent manner. We also assessed the defense response against a variety of potato pathogens, such as protists (Phytophthora infestans and Spongospora subterranea) and fungi (Verticillium dahliae and Colletotrichum coccodes). Our results show that extracellular pH increases within 30 min in proportion to the number of pathogen spores added. Consistently with the alkalinization effect, the higher transcription level of several defense-related genes and production of reactive oxygen species was observed. Our results demonstrate that the alkalinization response is an effective marker to study early stages of defense response in potatoes.

Movement and nucleocapsid proteins coded by two tospovirus species interact through multiple binding regions in mixed infections.

Negative-stranded tospoviruses (family: Bunyaviridae) are among the most agronomically important viruses. Some of the tospoviruses are known to exist as mixed infections in the same host plant. Iris yellow spot virus (IYSV) and Tomato spotted wilt virus (TSWV) were used to study virus-virus interaction in dually infected host plants. Viral genes of both viruses were separately cloned into binary pSITE-BiFC vectors. BiFC results showed that the N and NSm proteins of IYSV interact with their counterparts coded by TSWV in dually infected Nicotiana benthamiana plants. BiFC results were further confirmed by pull down and yeast-2-hybrid (Y2H) assays. Interacting regions of the N and NSm proteins were also identified by Y2H system and ß-galactosidase activity. Several regions of the N and NSm were found interacting with each other. The regions involved in these interactions are presumed to be critical for the functioning of the tospovirus N and NSm proteins. This is the first report of in vivo protein interactions of distinct tospoviruses in mixed infection.

In vivo localization of iris yellow spot tospovirus (Bunyaviridae)-encoded proteins and identification of interacting regions of nucleocapsid and movement proteins.

BACKGROUND: Localization and interaction studies of viral proteins provide important information about their replication in their host plants. Tospoviruses (Family Bunyaviridae) are economically important viruses affecting numerous field and horticultural crops. Iris yellow spot virus (IYSV), one of the tospoviruses, has recently emerged as an important viral pathogen of Allium spp. in many parts of the world. We studied the in vivo localization and interaction patterns of the IYSV proteins in uninfected and infected Nicotiana benthamiana and identified the interacting partners. PRINCIPAL FINDINGS: Bimolecular fluorescence complementation (BiFC) analysis demonstrated homotypic and heterotypic interactions between IYSV nucleocapsid (N) and movement (NSm) proteins. These interactions were further confirmed by pull-down assays. Additionally, interacting regions of IYSV N and NSm were identified by the yeast-2-hybrid system and ß-galactosidase assay. The N protein self-association was found to be mediated through the N- and C-terminal regions making head to tail interaction. Self-interaction of IYSV NSm was shown to occur through multiple interacting regions. In yeast-2-hybrid assay, the N- and C-terminal regions of IYSV N protein interacted with an N-terminal region of IYSV NSm protein. CONCLUSION/SIGNIFICANCE: Our studies provide new insights into localization and interactions of IYSV N and NSm proteins. Molecular basis of these interactions was studied and is discussed in the context of tospovirus assembly, replication, and infection processes.

SABP2, a methyl salicylate esterase is required for the systemic acquired resistance induced by acibenzolar-S-methyl in plants.

Tobacco SABP2, a 29kDa protein catalyzes the conversion of methyl salicylic acid (MeSA) into salicylic acid (SA) to induce SAR. Pretreatment of plants with acibenzolar-S-methyl (ASM), a functional analog of salicylic acid induces systemic acquired resistance (SAR). Data presented in this paper suggest that SABP2 catalyzes the conversion of ASM into acibenzolar to induce SAR. Transgenic SABP2-silenced tobacco plants when treated with ASM, fail to express PR-1 proteins and do not induce robust SAR expression. When treated with acibenzolar, full SAR is induced in SABP2-silenced plants. These results show that functional SABP2 is required for ASM-mediated induction of resistance.