XAP5 CIRCADIAN TIMEKEEPER Affects Both DNA Damage Responses and Immune Signaling in Arabidopsis.

Numerous links have been reported between immune response and DNA damage repair pathways in both plants and animals but the precise nature of the relationship between these fundamental processes is not entirely clear. Here, we report that XAP5 CIRCADIAN TIMEKEEPER (XCT), a protein highly conserved across eukaryotes, acts as a negative regulator of immunity in Arabidopsis thaliana and plays a positive role in responses to DNA damaging radiation. We find xct mutants have enhanced resistance to infection by a virulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, and are hyper-responsive to the defense-activating hormone salicylic acid (SA) when compared to wild-type. Unlike most mutants with constitutive effector-triggered immunity (ETI), xct plants do not have increased levels of SA and retain enhanced immunity at elevated temperatures. Genetic analysis indicates XCT acts independently of NONEXPRESSOR OF PATHOGENESIS RELATED GENES1 (NPR1), which encodes a known SA receptor. Since DNA damage has been reported to potentiate immune responses, we next investigated the DNA damage response in our mutants. We found xct seedlings to be hypersensitive to UV-C and γ radiation and deficient in phosphorylation of the histone variant H2A.X, one of the earliest known responses to DNA damage. These data demonstrate that loss of XCT causes a defect in an early step of the DNA damage response pathway. Together, our data suggest that alterations in DNA damage response pathways may underlie the enhanced immunity seen in xct mutants.

The N-terminus of the cauliflower mosaic virus aphid transmission protein P2 is involved in transmission body formation and microtubule interaction.

Cauliflower mosaic virus (CaMV) is transmitted by aphids using the non-circulative transmission mode: when the insects feed on infected leaves, virus particles from infected cells attach rapidly to their stylets and are transmitted to a new host when the aphids change plants. Mandatory for CaMV transmission, the viral helper protein P2 mediates as a molecular linker binding of the virus particles to the aphid stylets. P2 is available in infected plant cells in a viral inclusion that is specialized for transmission and named the transmission body (TB). When puncturing an infected leaf cell, the aphid triggers an ultra-rapid viral response, necessary for virus acquisition and called transmission activation: The TB disrupts and P2 is redistributed onto cortical microtubules, together with virus particles that are simultaneously set free from virus factories and join P2 on the microtubules to form the so-called mixed networks (MNs). The MNs are the predominant structure from which CaMV is acquired by aphids. However, the P2 domains involved in microtubule interaction are not known. To identify P2 regions involved in its functions, we generated a set of P2 mutants by alanine scanning and analyzed them in the viral context for their capacity to form a TB, to interact with microtubules and to transmit CaMV. Our results show that contrary to the previously characterized P2-P2 and P2-virion binding sites in its C-terminus, the microtubule binding site is contained in the N-terminal half of P2. Further, this region is important for TB formation since some P2 mutant proteins did not accumulate in TBs but were retained in the viral factories where P2 is translated. Taken together, the N-terminus of P2 is not only involved in vector interaction as previously reported, but also in interaction with microtubules and in formation of TBs.

Optimizing the PBS1 Decoy System to Confer Resistance to Potyvirus Infection in Arabidopsis and Soybean.

The Arabidopsis resistance protein RPS5 is activated by proteolytic cleavage of the protein kinase PBS1 by the Pseudomonas syringae effector protease AvrPphB. We have previously shown that replacing seven amino acids at the cleavage site of PBS1 with a motif cleaved by the NIa protease of turnip mosaic virus (TuMV) enables RPS5 activation upon TuMV infection. However, this engineered resistance conferred a trailing necrosis phenotype indicative of a cell-death response too slow to contain the virus. We theorized this could result from a positional mismatch within the cell between PBS1TuMV, RPS5, and the NIa protease. To test this, we relocalized PBS1TuMV and RPS5 to cellular sites of NIa accumulation. These experiments revealed that relocation of RPS5 away from the plasma membrane compromised RPS5-dependent cell death in Nicotiana benthamiana, even though PBS1 was efficiently cleaved. As an alternative approach, we tested whether overexpression of plasma membrane-localized PBS1TuMV could enhance RPS5 activation by TuMV. Significantly, overexpressing the PBS1TuMV decoy protein conferred complete resistance to TuMV when delivered by either agrobacterium or by aphid transmission, showing that RPS5-mediated defense responses are effective against bacterial and viral pathogens. Lastly, we have now extended this PBS1 decoy approach to soybean by modifying a soybean PBS1 ortholog to be cleaved by the NIa protease of soybean mosaic virus (SMV). Transgenic overexpression of this soybean PBS1 decoy conferred immunity to SMV, demonstrating that we can use endogenous PBS1 proteins in crop plants to engineer economically relevant disease resistant traits.

A Non-Conserved p33 Protein of Citrus Tristeza Virus Interacts with Multiple Viral Partners.

The RNA genome of citrus tristeza virus (CTV), one of the most damaging viral pathogens of citrus, contains 12 open reading frames resulting in production of at least 19 proteins. Previous studies on the intraviral interactome of CTV revealed self-interaction of the viral RNA-dependent RNA polymerase, the major coat protein (CP), p20, p23, and p33 proteins, while heterologous interactions between the CTV proteins have not been characterized. In this work, we examined interactions between the p33 protein, a nonconserved protein of CTV, which performs multiple functions in the virus infection cycle and is needed for virus ability to infect the extended host range, with other CTV proteins shown to mediate virus interactions with its plant hosts. Using yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays, we demonstrated that p33 interacts with three viral proteins, i.e., CP, p20, and p23, in vivo and in planta. Coexpression of p33, which is an integral membrane protein, resulted in a shift in the localization of the p20 and p23 proteins toward the subcellular crude-membrane fraction. Upon CTV infection, the four proteins colocalized in the CTV replication factories. In addition, three of them, CP, p20, and p23, were found in the p33-formed membranous structures. Using bioinformatic analyses and mutagenesis, we found that the N-terminus of p33 is involved in the interactions with all three protein partners. A potential role of these interactions in virus ability to infect the extended host range is discussed.

A polerovirus, Potato leafroll virus, alters plant-vector interactions using three viral proteins.

Potato leafroll virus (PLRV), genus Polerovirus, family Luteoviridae, is a major pathogen of potato worldwide. PLRV is transmitted among host plants by aphids in a circulative-nonpropagative manner. Previous studies have demonstrated that PLRV infection increases aphid fecundity on, and attraction to, infected plants as compared to controls. However, the molecular mechanisms mediating this relationship are still poorly understood. In this study, we measured the impact of PLRV infection on plant-aphid interactions and plant chemistry in two hosts: Solanum tuberosum and Nicotiana benthamiana. Our study demonstrates that PLRV infection attenuates the induction of aphid-induced jasmonic acid and ethylene in S. tuberosum and N. benthamiana. Using transient expression experiments, insect bioassays and chemical analysis, we show that expression of three PLRV proteins (P0, P1, and P7) mediate changes in plant-aphid interactions and inhibition of aphid-induced jasmonic acid and ethylene in N. benthamiana. This study enhances our understanding of the plant-vector-pathogen interface by elucidating new mechanisms by which plant viruses transmitted in a circulative manner can manipulate plant hosts.

Multiplex quantitative PCR for single-reaction genetically modified (GM) plant detection and identification of false-positive GM plants linked to Cauliflower mosaic virus (CaMV) infection.

BACKGROUND: Most genetically modified (GM) plants contain a promoter, P35S, from the plant virus, Cauliflower mosaic virus (CaMV), and many have a terminator, TNOS, derived from the bacterium, Agrobacterium tumefaciens. Assays designed to detect GM plants often target the P35S and/or TNOS DNA sequences. However, because the P35S promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by this naturally-occurring virus. RESULTS: Here we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed quantitative PCR (qPCR) reaction. Following initial testing and optimization via PCR and singleplex-to-multiplex qPCR on both plasmid and plant DNA, TaqMan qPCR probes with different fluorescence wavelengths were designed to target actin (a positive-control plant gene), P35S, P3 (a CaMV-specific gene), and TNOS. We tested the specificity of our quadruplex qPCR assay using different DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection, and by using commercial and industrial samples. The limit of detection (LOD) of each target was determined to be 1% for actin, 0.001% for P35S, and 0.01% for both P3 and TNOS. CONCLUSIONS: This assay was able to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction for all samples tested in this study, suggesting that this protocol is broadly applicable and readily transferrable to any interested parties with a qPCR platform.

Ethylene signaling mediates potyvirus spread by aphid vectors.

Plant pathogens can influence host characteristics such as volatile emissions, nutrient composition or plant color, modulating vector and non-vector insect dynamics in the ecosystem. While previous research has focused on insect attraction and dispersal to infected plants, little is known about mechanisms mediating these interactions. Here, we investigate the role of ethylene in green peach aphid (Myzus persicae) attraction to potyvirus-infected plants. In our experiments, we utilized two different potyviruses, Potato virus Y (PVY) and Turnip mosaic virus, in lab and field experiments. Consistent with previous studies, we show that greater numbers of aphids settle on potyvirus-infected plants in the lab and greater numbers of aphids are found in PVY-infected potato (Solanum tuberosum) fields compared to controls. In laboratory experiments, inhibition of ethylene signaling in plants either chemically or genetically prevented aphids from preferentially settling on potyvirus-infected plants. Virus spread was reduced in lab arenas by over 80% when ethylene signaling was inhibited chemically. Despite this, ethylene inhibition had no significant impact on virus spread in field mesocosms. Our results indicate that induction of ethylene signaling by potyviruses mediates aphid attraction to infected plants and virus spread; however, additional factors may contribute to plant-vector dynamics in complex communities. Specific components of ethylene signaling may be important targets for future management of vector-borne viruses and research on mechanisms mediating plant-vector-virus interactions.

Network Analysis Reveals a Role for Salicylic Acid Pathway Components in Shade Avoidance.

Plants have sophisticated mechanisms for sensing neighbor shade. To maximize their ability to compete for light, plants respond to shade through enhanced elongation and physiological changes. The shade avoidance response affects many different organs and growth stages, yet the signaling pathways underlying this response have mostly been studied in seedlings. We assayed transcriptome changes in response to shade across a 2-d time course in the wild type and 12 Arabidopsis (Arabidopsis thaliana) mutants. The resulting temporal map of transcriptional responses to shade defines early and late responses in adult plants, enabling us to determine connections between key signaling genes and downstream responses. We found a pervasive and unexpectedly strong connection between shade avoidance and genes related to salicylic acid, suggesting salicylic acid signaling to be an important shade avoidance growth regulator. We tested this connection and found that several mutants disrupting salicylic acid levels or signaling were defective in shade avoidance. The effect of these mutations on shade avoidance was specific to petiole elongation; neither hypocotyl nor flowering time responses were altered, thereby defining important stage-specific differences in the downstream shade avoidance signaling pathway. Shade treatment did not change salicylic acid levels, indicating that the mediation of shade avoidance by salicylic acid is not dependent on the modulation of salicylic acid levels. These results demonstrate that salicylic acid pathway genes also are key components of petiole shade avoidance.

The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the Nucleus and Regulates Defense Hormone Expression during Plant Innate Immunity.

Plants employ cell-surface pattern recognition receptors (PRRs) to detect pathogens. Although phytohormones produced during PRR signaling play an essential role in innate immunity, a direct link between PRR activation and hormone regulation is unknown. EFR is a PRR that recognizes bacterial EF-Tu and activates immune signaling. Here we report that EFR regulates the phytohormone jasmonic acid (JA) through direct phosphorylation of a receptor-like cytoplasmic kinase, BIK1. The BIK1 structure revealed that the EFR-phosphorylated sites reside on a uniquely extended loop away from the BIK1 kinase core domain. Phosphomimetic mutations of these sites resulted in increased phytohormones and enhanced resistance to bacterial infections. In addition to its documented plasma membrane localization, BIK1 also localizes to the nucleus and interacts directly with WRKY transcription factors involved in the JA and salicylic acid (SA) regulation. These findings demonstrate the mechanistic basis of signal transduction from PRR to phytohormones, mediated through a PRR-BIK1-WRKY axis.

A viral protease relocalizes in the presence of the vector to promote vector performance.

Vector-borne pathogens influence host characteristics relevant to host-vector contact, increasing pathogen transmission and survival. Previously, we demonstrated that infection with Turnip mosaic virus, a member of one of the largest families of plant-infecting viruses, increases vector attraction and reproduction on infected hosts. These changes were due to a single viral protein, NIa-Pro. Here we show that NIa-Pro responds to the presence of the aphid vector during infection by relocalizing to the vacuole. Remarkably, vacuolar localization is required for NIa-Pro's ability to enhance aphid reproduction on host plants, vacuole localization disappears when aphids are removed, and this phenomenon occurs for another potyvirus, Potato virus Y, suggesting a conserved role for the protein in vector-host interactions. Taken together, these results suggest that potyviruses dynamically respond to the presence of their vectors, promoting insect performance and transmission only when needed.

Simultaneous visualization of two Citrus tristeza virus genotypes provides new insights into the structure of multi-component virus populations in a host.

Complex Citrus tristeza virus (CTV) populations composed of mixtures of different strains of the virus are commonly found in citrus trees in the field. At present, little is known about how these populations are formed, maintained, and how they are structured within a host. Here we used a novel in situ hybridization approach allowing simultaneous visualization of two different RNA targets with high sensitivity and specificity to examine the distribution of two isolates, T36 and T68-1, representing phylogenetically distinct strains of CTV, in a citrus host in single and mixed infections. Remarkably, in doubly inoculated plants the two virus variants appeared to be well mixed within the infected tissue and showed no spatial segregation. In addition, both CTV variants were often found occupying the same cells. Possible mechanisms involved in shaping CTV populations and the biological significance of the observed lack of structural separation of the individual components are discussed.

The conundrum of a unique protein encoded by citrus tristeza virus that is dispensable for infection of most hosts yet shows characteristics of a viral movement protein.

Citrus tristeza virus (CTV), one of the most economically important viruses, produces a unique protein, p33, which is encoded only in the genomes of isolates of CTV. Recently, we demonstrated that membrane association of the p33 protein confers virus ability to extend its host range. In this work we show that p33 shares characteristics of viral movement proteins. Upon expression in a host cell, the protein localizes to plasmodesmata and displays the ability to form extracellular tubules. Furthermore, p33 appears to traffic via the cellular secretory pathway and the actin network to plasmodesmata locations and is likely being recycled through the endocytic pathway. Finally, our study reveals that p33 colocalizes with a putative movement protein of CTV, the p6 protein. These results suggest a potential role of p33 as a noncanonical viral movement protein, which mediates virus translocation in the specific hosts.

Disruption of Ethylene Responses by Turnip mosaic virus Mediates Suppression of Plant Defense against the Green Peach Aphid Vector.

Plants employ diverse responses mediated by phytohormones to defend themselves against pathogens and herbivores. Adapted pathogens and herbivores often manipulate these responses to their benefit. Previously, we demonstrated that Turnip mosaic virus (TuMV) infection suppresses callose deposition, an important plant defense induced in response to feeding by its aphid vector, the green peach aphid (Myzus persicae), and increases aphid fecundity compared with uninfected control plants. Further, we determined that production of a single TuMV protein, Nuclear Inclusion a-Protease (NIa-Pro) domain, was responsible for changes in host plant physiology and increased green peach aphid reproduction. To characterize the underlying molecular mechanisms of this phenomenon, we examined the role of three phytohormone signaling pathways, jasmonic acid, salicylic acid, and ethylene (ET), in TuMV-infected Arabidopsis (Arabidopsis thaliana), with or without aphid herbivory. Experiments with Arabidopsis mutants ethylene insensitive2 and ethylene response1, and chemical inhibitors of ET synthesis and perception (aminoethoxyvinyl-glycine and 1-methylcyclopropene, respectively), show that the ET signaling pathway is required for TuMV-mediated suppression of Arabidopsis resistance to the green peach aphid. Additionally, transgenic expression of NIa-Pro in Arabidopsis alters ET responses and suppresses aphid-induced callose formation in an ET-dependent manner. Thus, disruption of ET responses in plants is an additional function of NIa-Pro, a highly conserved potyvirus protein. Virus-induced changes in ET responses may mediate vector-plant interactions more broadly and thus represent a conserved mechanism for increasing transmission by insect vectors across generations.

Membrane association of a nonconserved viral protein confers virus ability to extend its host range.

Citrus tristeza virus (CTV), the largest and most complex member of the family Closteroviridae, encodes a unique protein, p33, which shows no homology with other known proteins, however, plays an important role in virus pathogenesis. In this study, we examined some of the characteristics of p33. We show that p33 is a membrane-associated protein that is inserted into the membrane via a transmembrane helix formed by hydrophobic amino acid residues at the C-terminal end of the protein. Removal of this transmembrane domain (TMD) dramatically altered the intracellular localization of p33. Moreover, the TMD alone was sufficient to confer membrane localization of an unrelated protein. Finally, a CTV variant that produced a truncated p33 lacking the TMD was unable to infect sour orange, one of the selected virus hosts, which infection requires p33, suggesting that membrane association of p33 is important for the ability of CTV to extend its host range.

Meloidogyne incognita - rice (Oryza sativa) interaction: a new model system to study plant-root-knot nematode interactions in monocotyledons.

BACKGROUND: Plant-parasitic nematodes developed strategies to invade and colonize their host plants, including expression of immune suppressors to overcome host defenses. Meloidogyne graminicola and M. incognita are root-knot nematode (RKN) species reported to damage rice (Oryza sativa L.) cultivated in upland and irrigated systems. Despite M. incognita wide host range, study of the molecular plant - RKN interaction has been so far limited to a few dicotyledonous model plants. The aim of this study was to investigate if the rice cv. Nipponbare widely used in rice genomic studies could be used as a suitable monocotyledon host plant for studying M. incognita pathogenicity mechanisms. Here we compared the ability of M. graminicola and M. incognita to develop and reproduce in Nipponbare roots. Next, we tested if RKNs modulates rice immunity-related genes expression in galls during infection and express the Mi-crt gene encoding an immune suppressor. RESULTS: Root galling, mature females, eggs and newly formed J2s nematodes were obtained for both species in rice cultivated in hydroponic culture system after 4-5 weeks. Meloidogyne graminicola reproduced at higher rates than M. incognita on Nipponbare and the timing of infection was shorter. In contrast, the infection characteristics compared by histological analysis were similar for both nematode species. Giant cells formed from 2 days after infection (DAI) with M. graminicola and from 6 DAI with M. incognita. Real-time PCR (qRT-PCR) data indicated that RKNs are able to suppress transcription of immune regulators genes, such as OsEDS1, OsPAD4 and OsWRKY13 in young galls. Four M. incognita reference genes (Mi-eif-3, Mi-GDP-2, Mi-Y45F10D.4, and Mi-actin) were selected for normalizing nematode gene expression studies in planta and in pre-parasitic J2s. Meloidogyne incognita expressed the immune suppressor calreticulin gene (Mi-crt) in rice roots all along its infection cycle. CONCLUSION: RKNs repress the transcription of key immune regulators in rice, likely in order to lower basal defence in newly-formed galls. The calreticulin Mi-CRT can be one of the immune-modulator effectors secreted by M. incognita in rice root tissues. Together, these data show that rice is a well suited model system to study host- M. incognita molecular interactions in monocotyledons.

Multiples fonctions des usines virales : l'exemple du virus de la mosaïque du chou- fleur (Cauliflower mosaic virus).

Many viruses form inclusion bodies in infected plant and mammalian cells. Their formation often requires membrane rearrangement of various organelles, but some inclusions form in the cytoplasm independently of the endomembrane system. In the latter case, they may resemble aggresomes or stress bodies but many inclusions do not seem to be related to any cellular structures. Synthesis, composition and size of these inclusions change with virus species. The best characterized inclusions create a "viral organelle" protecting viruses from host defenses and optimizing viral replication and assembly. These inclusions are also called viral factories. Recently, more complex and original functions were described for viral factories. This is exemplified here for Cauliflower mosaic virus (CaMV) factories. Unexpectedly, besides replication, CaMV factories also participate in another crucial step of the viral cycle: vector-transmission by aphids.

Virus factories of cauliflower mosaic virus are virion reservoirs that engage actively in vector transmission.

Cauliflower mosaic virus (CaMV) forms two types of inclusion bodies within infected plant cells: numerous virus factories, which are the sites for viral replication and virion assembly, and a single transmission body (TB), which is specialized for virus transmission by aphid vectors. The TB reacts within seconds to aphid feeding on the host plant by total disruption and redistribution of its principal component, the viral transmission helper protein P2, onto microtubules throughout the cell. At the same time, virions also associate with microtubules. This redistribution of P2 and virions facilitates transmission and is reversible; the TB reforms within minutes after vector departure. Although some virions are present in the TB before disruption, their subsequent massive accumulation on the microtubule network suggests that they also are released from virus factories. Using drug treatments, mutant viruses, and exogenous supply of viral components to infected protoplasts, we show that virions can rapidly exit virus factories and, once in the cytoplasm, accumulate together with the helper protein P2 on the microtubule network. Moreover, we show that during reversion of this phenomenon, virions from the microtubule network can either be incorporated into the reverted TB or return to the virus factories. Our results suggest that CaMV factories are dynamic structures that participate in vector transmission by controlled release and uptake of virions during TB reaction.

Early interactions during the encounter of plants, aphids and arboviruses.

Aphids infest many plants and cause damage by depriving them of nutrients and by transmitting many viral diseases. Aphid infestation and arbovirus transmission are controlled by establishment (or not) of a compatible reaction between the insects and the plants. This reaction is the result of defense reactions of the plant and counter-defense reactions of the parasite. Contrarily to plant-bacteria, plant-fungi and plant-herbivorous insects pathosystems, the plant-aphid pathosystem is understudied, although recent advances have begun to uncover some of its details. Especially the very early steps in plant-aphid interactions are hardly known. We here resume the present knowledge of these interactions. We discuss further how an aphid-transmitted plant virus that is transmitted during the first moments of the plant-aphid encounter, might help to study the very early plant aphid interactions.

A virus responds instantly to the presence of the vector on the host and forms transmission morphs.

Many plant and animal viruses are spread by insect vectors. Cauliflower mosaic virus (CaMV) is aphid-transmitted, with the virus being taken up from specialized transmission bodies (TB) formed within infected plant cells. However, the precise events during TB-mediated virus acquisition by aphids are unknown. Here, we show that TBs react instantly to the presence of the vector by ultra-rapid and reversible redistribution of their key components onto microtubules throughout the cell. Enhancing or inhibiting this TB reaction pharmacologically or by using a mutant virus enhanced or inhibited transmission, respectively, confirming its requirement for efficient virus-acquisition. Our results suggest that CaMV can perceive aphid vectors, either directly or indirectly by sharing the host perception. This novel concept in virology, where viruses respond directly or via the host to the outside world, opens new research horizons, that is, investigating the impact of 'perceptive behaviors' on other steps of the infection cycle.DOI:http://dx.doi.org/10.7554/eLife.00183.001.

Host cell processes to accomplish mechanical and non-circulative virus transmission.

Mechanical vector-less transmission of viruses, as well as vector-mediated non-circulative virus transmission, where the virus attaches only to the exterior of the vector during the passage to a new host, are apparently simple processes: the viruses are carried along with the wind, the food or by the vector to a new host. We discuss here, using the examples of the non-circulatively transmitted Cauliflower mosaic virus that binds to its aphid vector's exterior mouthparts, and that of the mechanically (during feeding activity) transmitted Autographa californica multicapsid nucleopolyhedrovirus, that transmission of these viruses is not so simple as previously thought. Rather, these viruses prepare their transmission carefully and long before the actual acquisition event. Host-virus interactions play a pivotal and specialised role in the future encounter with the vector or the new host. This ensures optimal propagation and enlarges the tremendous bottleneck transmission presents for viruses and other pathogens.