Modulation of 1O2-mediated retrograde signaling by the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein, a central integrator of stress and energy signaling

Shortly after the release of singlet oxygen (¹O₂) in chloroplasts, changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Extensive genetic screens aimed at identifying constituents involved in ¹O₂-mediated plastid-to-nucleus signaling have failed to identify extraplastidic signaling components. This finding suggests that ¹O₂-mediated signals are not translocated to the nucleus via a single linear pathway, but rather through a signaling network that is difficult to block by single mutations. The complexity of this signaling network has been tackled by mutagenizing a transgenic flu line expressing the luciferase reporter gene under the control of the promoter of a ¹O₂-responsive AAA-ATPase gene (At3g28580) and isolating second site mutants that constitutively express the reporter gene at a high level. One of the mutants was shown by map-based cloning and sequencing to contain a single amino acid change in the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein. PRL1 suppresses the expression of AAA-ATPase and other ¹O₂-responsive genes. PRL1 seems to play a major role in modulating responses of plants to environmental changes by interconnecting ¹O₂-mediated retrograde signaling with other signaling pathways.     

Signaling pathways that regulate the enhanced disease resistance of Arabidopsis "defense, no death" mutants

Arabidopsis dnd1 and dnd2 mutants lack cyclic nucleotide-gated ion channel proteins and carry out avirulence or resistance gene-mediated defense with a greatly reduced hypersensitive response (HR). They also exhibit elevated broad-spectrum disease resistance and constitutively elevated salicylic acid (SA) levels. We examined the contributions of NPR1, SID2 (EDS16), NDR1, and EIN2 to dnd phenotypes. Mutations that affect SA accumulation or signaling (sid2, npr1, and ndr1) abolished the enhanced resistance of dnd mutants against Pseudomonas syringae pv. tomato and Hyaloperonospora parasitica but not Botrytis cinerea. When SA-associated pathways were disrupted, the constitutive activation of NPR1-dependent and NPR1-independent and SA-dependent pathways was redirected toward PDF1.2-associated pathways. This PDF1.2 overexpression was downregulated after infection by P. syringae. Disruption of ethylene signaling abolished the enhanced resistance to B. cinerea but not P. syringae or H. parasitica. However, loss of NPR1, SID2, NDR1, or EIN2 did not detectably alter the reduced HR in dnd mutants. The susceptibility of dnd ein2 plants to B. cinerea despite their reduced-HR phenotype suggests that cell death repression is not the primary cause of dnd resistance to necrotrophic pathogens. The partial restoration of resistance to B. cinerea in dnd1 npr1 ein2 triple mutants indicated that this resistance is not entirely EIN2 dependent. The above findings indicate that the broad-spectrum resistance of dnd mutants occurs due to activation or sensitization of multiple defense pathways, yet none of the investigated pathways are required for the reduced-HR phenotype.     

The U-Box/ARM E3 ligase PUB13 regulates cell death, defense, and flowering time in Arabidopsis

The components in plant signal transduction pathways are intertwined and affect each other to coordinate plant growth, development, and defenses to stresses. The role of ubiquitination in connecting these pathways, particularly plant innate immunity and flowering, is largely unknown. Here, we report the dual roles for the Arabidopsis (Arabidopsis thaliana) Plant U-box protein13 (PUB13) in defense and flowering time control. In vitro ubiquitination assays indicated that PUB13 is an active E3 ubiquitin ligase and that the intact U-box domain is required for the E3 ligase activity. Disruption of the PUB13 gene by T-DNA insertion results in spontaneous cell death, the accumulation of hydrogen peroxide and salicylic acid (SA), and elevated resistance to biotrophic pathogens but increased susceptibility to necrotrophic pathogens. The cell death, hydrogen peroxide accumulation, and resistance to necrotrophic pathogens in pub13 are enhanced when plants are pretreated with high humidity. Importantly, pub13 also shows early flowering under middle- and long-day conditions, in which the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 and FLOWERING LOCUS T is induced while FLOWERING LOCUS C expression is suppressed. Finally, we found that two components involved in the SA-mediated signaling pathway, SID2 and PAD4, are required for the defense and flowering-time phenotypes caused by the loss of function of PUB13. Taken together, our data demonstrate that PUB13 acts as an important node connecting SA-dependent defense signaling and flowering time regulation in Arabidopsis.     

Multiple roles of WIN3 in regulating disease resistance, cell death, and flowering time in Arabidopsis

The salicylic acid (SA) regulatory gene HOPW1-1-INTERACTING3 (WIN3) was previously shown to confer resistance to the biotrophic pathogen Pseudomonas syringae. Here, we report that WIN3 controls broad-spectrum disease resistance to the necrotrophic pathogen Botrytis cinerea and contributes to basal defense induced by flg22, a 22-amino acid peptide derived from the conserved region of bacterial flagellin proteins. Genetic analysis indicates that WIN3 acts additively with several known SA regulators, including PHYTOALEXIN DEFICIENT4, NONEXPRESSOR OF PR GENES1 (NPR1), and SA INDUCTION-DEFICIENT2, in regulating SA accumulation, cell death, and/or disease resistance in the Arabidopsis (Arabidopsis thaliana) mutant acd6-1. Interestingly, expression of WIN3 is also dependent on these SA regulators and can be activated by cell death, suggesting that WIN3-mediated signaling is interconnected with those derived from other SA regulators and cell death. Surprisingly, we found that WIN3 and NPR1 synergistically affect flowering time via influencing the expression of flowering regulatory genes FLOWERING LOCUS C and FLOWERING LOCUS T. Taken together, our data reveal that WIN3 represents a novel node in the SA signaling networks to regulate plant defense and flowering time. They also highlight that plant innate immunity and development are closely connected processes, precise regulation of which should be important for the fitness of plants.     

The evolutionarily conserved MOS4-associated complex

Recent work in plant immunity has shown that MOS4, a known intermediate in R protein mediated resistance, is a core member of the nuclear MOS4-associated complex (MAC). This complex is highly conserved in eukaryotes, as orthologous complexes known as the CDC5L-SNEV^Prp19-Pso4 complex and the Nineteen complex (NTC) were previously identified in human and yeast, respectively. The involvement of these complexes in pre-mRNA splicing and spliceosome assembly suggests that the MAC probably has a similar function in plants. Double mutants of any two MAC components are lethal, whereas single mutants of the MAC core components mos4, Atcdc5, mac3, and prl1 are all viable and display pleiotropic defects. This suggests that while the MAC is required for some essential biological function such as splicing, individual MAC components are not crucial for complex functionality and likely have regulatory roles in other biological processes such as plant immunity and flowering time control. Future studies on MAC components in Arabidopsis will provide further insight into the regulatory mechanisms of the MAC on specific biological processes.     

Heterotrimeric G proteins facilitate Arabidopsis resistance to necrotrophic pathogens and are involved in jasmonate signaling

Heterotrimeric G proteins have been previously linked to plant defense; however a role for the Gbetagamma dimer in defense signaling has not been described to date. Using available Arabidopsis (Arabidopsis thaliana) mutants lacking functional Galpha or Gbeta subunits, we show that defense against the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum is impaired in Gbeta-deficient mutants while Galpha-deficient mutants show slightly increased resistance compared to wild-type Columbia ecotype plants. In contrast, responses to virulent (DC3000) and avirulent (JL1065) strains of Pseudomonas syringae appear to be independent of heterotrimeric G proteins. The induction of a number of defense-related genes in Gbeta-deficient mutants were severely reduced in response to A. brassicicola infection. In addition, Gbeta-deficient mutants exhibit decreased sensitivity to a number of methyl jasmonate-induced responses such as induction of the plant defensin gene PDF1.2, inhibition of root elongation, seed germination, and growth of plants in sublethal concentrations of methyl jasmonate. In all cases, the behavior of the Galpha-deficient mutants is coherent with the classic heterotrimeric mechanism of action, indicating that jasmonic acid signaling is influenced by the Gbetagamma functional subunit but not by Galpha. We hypothesize that Gbetagamma acts as a direct or indirect enhancer of the jasmonate signaling pathway in plants.     

Arabidopsis enhanced disease susceptibility mutants exhibit enhanced susceptibility to several bacterial pathogens and alterations in PR-1 gene expression.

To identify plant defense responses that limit pathogen attack, Arabidopsis eds mutants that exhibit enhanced disease susceptibility to the virulent bacterial pathogen Pseudomonas syringae pv maculicola ES4326 were previously identified. In this study, we show that each of four eds mutants (eds5-1, eds6-1, eds7-1, and eds9-1) has a distinguishable phenotype with respect to the degree of susceptibility to a panel of bacterial phytopathogens and the ability to activate pathogenesis-related PR-1 gene expression after pathogen attack. None of the four eds mutants exhibited observable defects in mounting a hypersensitive response. Although all four eds mutants were also capable of mounting a systemic acquired resistance response, enhanced growth of P. s. maculicola ES4326 was still apparent in the secondarily infected leaves of three of the eds mutants. These data indicate that eds genes define a diverse set of previously unknown defense responses that affect resistance to virulent pathogens.     

Positive and negative regulation of salicylic acid-dependent cell death and pathogen resistance in Arabidopsis lsd6 and ssi1 mutants

Salicylic acid (SA) is a key defense molecule in higher plants that is required for resistance to diverse pathogens. A number of mutants of Arabidopsis with elevated resistance to pathogens and constitutive activation of defense-related genes and cell death have been shown to require SA for all of their phenotypes. These mutants potentially identify interesting regulatory genes that control diverse SA responses. When dominant mutations confer SA-dependent phenotypes, it is important to know the genetic basis of dominance in order to draw conclusions on the possible mechanisms of action of the genes identified. Here I characterize the basis of the dominant phenotypes conferred by the ssi1 and lsd6 mutations. I show that ssi1 is haploinsufficient, while lsd6 is a gain-of-function mutation. Thus, SA-dependent responses are under both negative and positive regulation.     

A deletion in NRT2.1 attenuates Pseudomonas syringae-induced hormonal perturbation, resulting in primed plant defenses

For an efficient defense response against pathogens, plants must coordinate rapid genetic reprogramming to produce an incompatible interaction. Nitrate Trasnporter2 (NRT2) gene family members are sentinels of nitrate availability. In this study, we present an additional role for NRT2.1 linked to plant resistance against pathogens. This gene antagonizes the priming of plant defenses against the bacterial pathogen Pseudomonas syringae pv tomato DC3000 (Pst). The nrt2 mutant (which is deficient in two genes, NRT2.1 and NRT2.2) displays reduced susceptibility to this bacterium. We demonstrate that modifying environmental conditions that stimulate the derepression of the NRT2.1 gene influences resistance to Pst independently of the total level of endogenous nitrogen. Additionally, hormonal homeostasis seemed to be affected in nrt2, which displays priming of salicylic acid signaling and concomitant irregular functioning of the jasmonic acid and abscisic acid pathways upon infection. Effector-triggered susceptibility and hormonal perturbation by the bacterium seem to be altered in nrt2, probably due to reduced sensitivity to the bacterial phytotoxin coronatine. The main genetic and metabolic targets of coronatine in Arabidopsis (Arabidopsis thaliana) remain largely unstimulated in nrt2 mutants. In addition, a P. syringae strain defective in coronatine synthesis showed the same virulence toward nrt2 as the coronatine-producing strain. Taken together, the reduced susceptibility of nrt2 mutants seems to be a combination of priming of salicylic acid-dependent defenses and reduced sensitivity to the bacterial effector coronatine. These results suggest additional functions for NRT2.1 that may influence plant disease resistance by down-regulating biotic stress defense mechanisms and favoring abiotic stress responses.     

The Ankyrin-Repeat Transmembrane Protein BDA1 Functions Downstream of the Receptor-Like Protein SNC2 to Regulate Plant Immunity

Plants utilize a large number of immune receptors to recognize pathogens and activate defense responses. A small number of these receptors belong to the receptor-like protein family. Previously, we showed that a gain-of-function mutation in the receptor-like protein SNC2 (for Suppressor of NPR1, Constitutive2) leads to constitutive activation of defense responses in snc2-1D mutant plants. To identify defense signaling components downstream of SNC2, we carried out a suppressor screen in the snc2-1D mutant background of Arabidopsis (Arabidopsis thaliana). Map-based cloning of one of the suppressor genes, BDA1 (for bian da; "becoming big" in Chinese), showed that it encodes a protein with amino-terminal ankyrin repeats and carboxyl-terminal transmembrane domains. Loss-of-function mutations in BDA1 suppress the dwarf morphology and constitutive defense responses in snc2-1D npr1-1 (for nonexpressor of pathogenesis-related genes1,1) and also result in enhanced susceptibility to bacterial pathogens. In contrast, a gain-of-function allele of bda1 isolated from a separate genetic screen to search for mutants with enhanced pathogen resistance was found to constitutively activate cell death and defense responses. These data suggest that BDA1 is a critical signaling component that functions downstream of SNC2 to regulate plant immunity.     

Components of Arabidopsis defense- and ethylene-signaling pathways regulate susceptibility to Cauliflower mosaic virus by restricting long-distance movement

We analyzed the susceptibility of Arabidopsis mutants with defects in salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signaling to infection by Cauliflower mosaic virus (CaMV). Mutants cpr1-1 and cpr5-2, in which SA-dependent defense signaling is activated constitutively, were substantially more resistant than the wild type to systemic infection, implicating SA signaling in defense against CaMV. However, SA-deficient NahG, sid2-2, eds5-1, and pad4-1 did not show enhanced susceptibility. A cpr5 eds5 double mutant also was resistant, suggesting that resistance in cpr5 may function partially independently of SA. Treatment of cpr5 and cpr5 eds5, but not cpr1, with salicyl-hydroxamic acid, an inhibitor of alternative oxidase, partially restored susceptibility to wild-type levels. Mutants etr1-1, etr1-3, and ein2-1, and two mutants with lesions in ET/JA-mediated defense, eds4 and eds8, also showed reduced virus susceptibility, demonstrating that ET-dependent responses also play a role in susceptibility. We used a green fluorescent protein (GFP)-expressing CaMV recombinant to monitor virus movement. In mutants with reduced susceptibility, cpr1-1, cpr5-2, and etr1-1, CaMV-GFP formed local lesions similar to the wild type, but systemic spread was almost completely absent in cpr1 and cpr5 and was substantially reduced in etr1-1. Thus, mutations with enhanced systemic acquired resistance or compromised ET signaling show diminished long-distance virus movement.     

Enhanced Disease Susceptibility 1 and Salicylic Acid Act Redundantly to Regulate Resistance Gene-Mediated Signaling

Resistance (R) protein–associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA) and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1), non–race-specific disease resistance 1 (NDR1), phytoalexin deficient 4 (PAD4), senescence associated gene 101 (SAG101), and EDS5, have been identified as components of resistance derived from many R proteins. Here, we show that EDS1 and SA fulfill redundant functions in defense signaling mediated by R proteins, which were thought to function independent of EDS1 and/or SA. Simultaneous mutations in EDS1 and the SA–synthesizing enzyme SID2 compromised hypersensitive response and/or resistance mediated by R proteins that contain coiled coil domains at their N-terminal ends. Furthermore, the expression of R genes and the associated defense signaling induced in response to a reduction in the level of oleic acid were also suppressed by compromising SA biosynthesis in the eds1 mutant background. The functional redundancy with SA was specific to EDS1. Results presented here redefine our understanding of the roles of EDS1 and SA in plant defense.     

Brush and spray: a high-throughput systemic acquired resistance assay suitable for large-scale genetic screening

Systemic acquired resistance (SAR) is a defense mechanism induced in the distal parts of plants after primary infection. It confers long-lasting protection against a broad spectrum of microbial pathogens. Lack of high-throughput assays has hampered the forward genetic analysis of SAR. Here, we report the development of an easy and efficient assay for SAR and its application in a forward genetic screen for SAR-deficient mutants in Arabidopsis (Arabidopsis thaliana). Using the new assay for SAR, we identified six flavin-dependent monooxygenase1, four AGD2-like defense response protein1, three salicylic acid induction-deficient2, one phytoalexin deficient4, and one avrPphB-susceptible3 alleles as well as a gain-of-function mutant of CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR3 designated camta3-3D. Like transgenic plants overexpressing CAMTA3, camta3-3D mutant plants exhibit compromised SAR and enhanced susceptibility to virulent pathogens, suggesting that CAMTA3 is a critical regulator of both basal resistance and SAR.     

Two putative RNA-binding proteins function with unequal genetic redundancy in the MOS4-associated complex

The MOS4-associated complex (MAC) is a highly conserved nuclear protein complex associated with the spliceosome. We recently purified the MAC from Arabidopsis (Arabidopsis thaliana) nuclei, identified its potential components by mass spectrometry, and showed that at least five core proteins in the MAC are required for defense responses in plants. Here, we report the characterization of a putative RNA-binding protein identified in the MAC named MAC5A and its close homolog MAC5B. We confirmed that MAC5A is a component of the MAC through coimmunoprecipitation with the previously described MAC protein CELL DIVISION CYCLE5 from Arabidopsis. In addition, like all other characterized MAC proteins, MAC5A fused to the Green Fluorescent Protein localizes to the nucleus. Double mutant analysis revealed that MAC5A and MAC5B are unequally redundant and that a double mac5a mac5b mutant results in lethality. Probably due to this partial redundancy, mac5a and mac5b single mutants do not exhibit enhanced susceptibility to virulent or avirulent pathogen infection. However, like other MAC mutations, mac5a-1 partially suppresses the autoimmune phenotypes of suppressor of npr1-1, constitutive1 (snc1), a gain-of-function mutant that expresses a deregulated Resistance protein. Our results suggest that MAC5A is a component of the MAC that contributes to snc1- mediated autoimmunity.     

A novel role for protein farnesylation in plant innate immunity

Plants utilize tightly regulated mechanisms to defend themselves against pathogens. Initial recognition results in activation of specific Resistance (R) proteins that trigger downstream immune responses, in which the signaling networks remain largely unknown. A point mutation in SUPPRESSOR OF NPR1 CONSTITUTIVE1 (SNC1), a RESISTANCE TO PERONOSPORA PARASITICA4 R gene homolog, renders plants constitutively resistant to virulent pathogens. Genetic suppressors of snc1 may carry mutations in genes encoding novel signaling components downstream of activated R proteins. One such suppressor was identified as a novel loss-of-function allele of ENHANCED RESPONSE TO ABSCISIC ACID1 (ERA1), which encodes the beta-subunit of protein farnesyltransferase. Protein farnesylation involves attachment of C15-prenyl residues to the carboxyl termini of specific target proteins. Mutant era1 plants display enhanced susceptibility to virulent bacterial and oomycete pathogens, implying a role for farnesylation in basal defense. In addition to its role in snc1-mediated resistance, era1 affects several other R-protein-mediated resistance responses against bacteria and oomycetes. ERA1 acts partly independent of abscisic acid and additively with the resistance regulator NON-EXPRESSOR OF PR GENES1 in the signaling network. Defects in geranylgeranyl transferase I, a protein modification similar to farnesylation, do not affect resistance responses, indicating that farnesylation is most likely specifically required in plant defense signaling. Taken together, we present a novel role for farnesyltransferase in plant-pathogen interactions, suggesting the importance of protein farnesylation, which contributes to the specificity and efficacy of signal transduction events.     

Characterization of Arabidopsis and rice DWD proteins and their roles as substrate receptors for CUL4-RING E3 ubiquitin ligases

A subset of WD40 proteins that contain a DWD motif (for DDB1 binding WD40) is reported to act as substrate receptors for DDB1-CUL4-ROC1 (for Damaged DNA Binding 1-Cullin 4-Regulator of Cullins 1) based E3 ubiquitin ligases in humans. Here, we report 85 Arabidopsis thaliana and 78 rice (Oryza sativa) proteins containing the conserved 16-amino acid DWD motif. We show by yeast two-hybrid and in vivo coimmunoprecipitation that 11 Arabidopsis DWD proteins directly interact with DDB1 and thus may serve as substrate receptors for the DDB1-CUL4 machinery. We further examine whether the DWD protein PRL1 (for Pleiotropic Regulatory Locus 1) may act as part of a CUL4-based E3 ligase. PRL1 directly interacts with DDB1, and prl1 and cul4cs mutants exhibited similar phenotypes, including altered responses to a variety of stimuli. Moreover, AKIN10 (for Arabidopsis SNF1 Kinase Homolog 10) was degraded more slowly in cell extracts of prl1 and cul4cs than in cell extracts of the wild type. Thus, both genetic and biochemical analyses support the conclusion that PRL1 is the substrate receptor of a CUL4-ROC1-DDB1-PRL1 E3 ligase involved in the degradation of AKIN10. This work adds a large new family to the current portfolio of plant E3 ubiquitin ligases.