Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector.

Plant innate immunity relies on the recognition of pathogen effector molecules by nucleotide-binding-leucine-rich repeat (NB-LRR) immune receptor families. Previously we have shown the N immune receptor, a member of TIR-NB-LRR family, indirectly recognizes the 50 kDa helicase (p50) domain of Tobacco mosaic virus (TMV) through its TIR domain. We have identified an N receptor-interacting protein, NRIP1, that directly interacts with both N's TIR domain and p50. NRIP1 is a functional rhodanese sulfurtransferase and is required for N to provide complete resistance to TMV. Interestingly, NRIP1 that normally localizes to the chloroplasts is recruited to the cytoplasm and nucleus by the p50 effector. As a consequence, NRIP1 interacts with N only in the presence of the p50 effector. Our findings show that a chloroplastic protein is intimately involved in pathogen recognition. We propose that N's activation requires a prerecognition complex containing the p50 effector and NRIP1.

Special delivery for MHC II via autophagy.

In this issue of Immunity, Blanchet et al. (2010) report that human immunodeficiency virus-1 inhibits macroautophagy in dendritic cells, attenuating MHC II presentation. Lee et al. (2010) previously revealed the requirement of autophagic machinery for MHC II presentation of herpes viral antigens.

Differential processing of Arabidopsis ubiquitin-like Atg8 autophagy proteins by Atg4 cysteine proteases.

Autophagy is a highly conserved biological process during which double membrane bound autophagosomes carry intracellular cargo material to the vacuole or lysosome for degradation and/or recycling. Autophagosome biogenesis requires Autophagy 4 (Atg4) cysteine protease-mediated processing of ubiquitin-like Atg8 proteins. Unlike single Atg4 and Atg8 genes in yeast, the Arabidopsis genome contains two Atg4 (AtAtg4a and AtAtg4b) and nine Atg8 (AtAtg8a-AtAtg8i) genes. However, we know very little about specificity of different AtAtg4s for processing of different AtAtg8s. Here, we describe a unique bioluminescence resonance energy transfer-based AtAtg8 synthetic substrate to assess AtAtg4 activity in vitro and in vivo. In addition, we developed a unique native gel assay of superhRLUC catalytic activity assay to monitor cleavage of AtAtg8s in vitro. Our results indicate that AtAtg4a is the predominant protease and that it processes AtAtg8a, AtAtg8c, AtAtg8d, and AtAtg8i better than AtAtg4b in vitro. In addition, kinetic analyses indicate that although both AtAtg4s have similar substrate affinity, AtAtg4a is more active than AtAtg4b in vitro. Activity of AtAtg4s is reversibly inhibited in vitro by reactive oxygen species such as H2O2. Our in vivo bioluminescence resonance energy transfer analyses in Arabidopsis transgenic plants indicate that the AtAtg8 synthetic substrate is efficiently processed and this is AtAtg4 dependent. These results indicate that the synthetic AtAtg8 substrate is used efficiently in the biogenesis of autophagosomes in vivo. Transgenic Arabidopsis plants expressing the AtAtg8 synthetic substrate will be a valuable tool to dissect autophagy processes and the role of autophagy during different biological processes in plants.

Autophagy and plant innate immunity: Defense through degradation.

Autophagy is a process of bulk degradation and nutrient sequestration that occurs in all eukaryotes. In plants, autophagy is activated during development, environmental stress, starvation, and senescence. Recent evidence suggests that autophagy is also necessary for the proper regulation of hypersensitive response programmed cell death (HR-PCD) during the plant innate immune response. We review autophagy in plants with emphasis on the role of autophagy during innate immunity. We hypothesize a role for autophagy in the degradation of pro-death signals during HR-PCD, with specific focus on reactive oxygen species and their sources. We propose that the plant chloroplasts are an important source of pro-death signals during HR-PCD, and that the chloroplast itself may be targeted for autophagosomal degradation by a process called chlorophagy.

All hands on deck­the role of chloroplasts, endoplasmic reticulum, and the nucleus in driving plant innate immunity.

Plant innate immunity is mediated by cell membrane and intracellular immune receptors that function in distinct and overlapping cell-signaling pathways to activate defense responses. It is becoming increasingly evident that immune receptors rely on components from multiple organelles for the generation of appropriate defense responses. This review analyzes the defense-related functions of the chloroplast, nucleus, and endoplasmic reticulum (ER) during plant innate immunity. It details the role of the chloroplasts in synthesizing defense-specific second messengers and discusses the retrograde signal transduction pathways that exist between the chloroplast and nucleus. Because the activities of immune modulators are regulated, in part, by their subcellular localization, the review places special emphasis on the dynamics and nuclear–cytoplasmic transport of immune receptors and regulators and highlights the importance of this process in generating orderly events during an innate immune response. The review also covers the recently discovered contributions of the ER quality-control pathways in ensuring the signaling competency of cell surface immune receptors or immune regulators.

Something old, something new: plant innate immunity and autophagy.

Autophagy performs a variety of established functions during plant growth and development. Recently, autophagy has been further implicated in the regulation of programmed cell death induced during the plant innate immune response. In this chapter we describe specific mechanisms through which autophagy may contribute to a successful defense against pathogen invasion. Accumulating evidence shows that the plant immune system utilizes the chloroplasts as primary sites for the regulation of cell death programs. Viruses also appear to utilize the chloroplast as a site of replication and accumulation, potentially inactivating chloroplast defense signaling in the process. Autophagy-like mechanisms have been observed to target the chloroplast, which we refer to as "chlorophagy," potentially targeting invasive viruses for degradation or regulating chloroplast-based signaling during the immune response. We hypothesize that chlorophagy is significant for the execution of plant immune defenses, during both basal and effector-triggered immunity.

The leucine-rich repeat domain in plant innate immunity: a wealth of possibilities.

The innate immune system of both plants and animals uses immune receptors to detect pathogens and trigger defence responses. Despite having distinct evolutionary origin, most plant and animal immune receptors have a leucine-rich repeat (LRR) domain. The LRR domain adopts a slender conformation that maximizes surface area and has been shown to be ideal for mediating protein-protein interactions. Although the LRR domain was expected to be a platform for pathogen recognition, the NB-LRR class of plant innate immune receptors uses its LRR domain to carry out many other roles. This review discusses the domain architecture of plant LRRs and the various roles ascribed to this motif.

P58(IPK), a plant ortholog of double-stranded RNA-dependent protein kinase PKR inhibitor, functions in viral pathogenesis.

P58(IPK) is a cellular inhibitor of the mammalian double-stranded RNA-activated protein kinase (PKR). Here we provide evidence for the existence of its homolog in plants and its role in viral infection at the organism level. Viral infection of P58(IPK)-silenced Nicotiana benthamiana and Arabidopsis knockouts leads to host death. This host cell death is associated with phosphorylation of the alpha subunit of eukaryotic translation initiation factor (eIF-2alpha). Loss of P58(IPK) leads to reduced virus titer, suggesting that wild-type P58(IPK) protein plays an important role in viral pathogenesis. Although our complementation results using mammalian P58(IPK) suggest conservation of the P58(IPK) pathway in plants and animals, its biological significance seems to be different in these two systems. In animals, P58(IPK) is recruited by the influenza virus to limit PKR-mediated innate antiviral response. In plants, P58(IPK) is required by viruses for virulence and therefore functions as a susceptibility factor.

Signaling in plant-microbe interactions.

Analysis of viral and bacterial pathogenesis has revealed common themes in the ways in which plants and animals respond to pathogenic agents. Pathogenic bacteria use macromolecule delivery systems (types III and IV) to deliver microbial avirulence proteins and transfer DNA-protein complexes directly into plant cells. The molecular events that constitute critical steps of plant-pathogen interactions seem to involve ligand-receptor mechanisms for pathogen recognition and the induction of signal transduction pathways in the plant that lead to defense responses. Unraveling the molecular basis of disease resistance pathways has laid a foundation for the rational design of crop protection strategies.

Autophagy in the control of programmed cell death.

Programmed cell death (PCD) is essential for plant development and immunity. Localized PCD is associated with the hypersensitive response (HR), which is a constituent of a successful plant innate immune response. Plants have developed mechanisms to meticulously prevent HR-PCD lesions from spreading. Our understanding of these mechanisms is still in its incipient stages. A recent study demonstrated that autophagy, a universally conserved process of macromolecule turnover, plays a pivotal role in controlling HR-PCD. The molecular identity of the mediators between the PCD and HR pathways is still obscure, but recent work has begun to shed light on the relationship between HR-PCD and autophagy and to suggest possible mechanisms for the regulation of these pathways.

Virus-induced gene silencing as a tool to identify host genes affecting viral pathogenicity.

Host factors are crucial determinants of viral pathogenicity. Identifying host factors and their contributions to virus infections may lead to the development of novel antiviral strategies. The recently developed virus-induced gene silencing (VIGS) approach offers a rapid means to knock down expression of a given gene in plants. VIGS can be used to determine biological function of candidate genes or to discover new genes that play a role in a given biological pathway. Here, we describe genome-wide Tobacco rattle virus (TRV)-based VIGS screening methods to identify host factors involved in viral pathogenicity.

A sequence required for -1 ribosomal frameshifting located four kilobases downstream of the frameshift site.

Programmed ribosomal frameshifting allows one mRNA to encode regulate expression of, multiple open reading frames (ORFs). The polymerase encoded by ORF 2 of Barley yellow dwarf virus (BYDV) is expressed via minus one (-1) frameshifting from the overlapping ORF 1. Previously, this appeared to be mediated by a 116 nt RNA sequence that contains canonical -1 frameshift signals including a shifty heptanucleotide followed by a highly structured region. However, unlike known -1 frameshift signals, the reporter system required the zero frame stop codon and did not require a consensus shifty site for expression of the -1 ORF. In contrast, full-length viral RNA required a functional shifty site for frameshifting in wheat germ extract, while the stop codon was not required. Increasing translation initiation efficiency by addition of a 5' cap on the naturally uncapped viral RNA, decreased the frameshift rate. Unlike any other known RNA, a region four kilobases downstream of the frameshift site was required for frameshifting. This included an essential 55 base tract followed by a 179 base tract that contributed to full frameshifting. The effects of most mutations on frameshifting correlated with the ability of viral RNA to replicate in oat protoplasts, indicating that the wheat germ extract accurately reflected control of BYDV RNA translation in the infected cell. However, the overall frameshift rate appeared to be higher in infected cells, based on immunodetection of viral proteins. These findings show that use of short recoding sequences out of context in reporter constructs may overlook distant signals. Most importantly, the remarkably long-distance interaction reported here suggests the presence of a novel structure that can facilitate ribosomal frameshifting.

Translational frameshifting by barley yellow dwarf virus RNA (PAV serotype) in Escherichia coli and in eukaryotic cell-free extracts.

The open reading frame (39K ORF) at the 5' end of the genome of barley yellow dwarf virus, PAV serotype (BYDV-PAV), overlaps with a 60K ORF by 13 nucleotides. Several approaches were used to show that the 60K ORF (putative polymerase gene) is translated by a low-frequency frameshift event in which some ribosomes shift into the 60K ORF rather than terminate at the 39K ORF stop codon. A sequence encompassing this region of overlap induced minus one (-1) translational frameshifting in heterologous and native contexts. In Escherichia coli, with the alpha subunit of lacZ used as a reporter gene, the rate of frameshifting caused by the BYDV-PAV sequence was approximately 3%. Amino acid sequencing of the transframe protein confirmed that ribosomes slip into the -1 frame in the overlapping region which includes a consensus shifty heptanucleotide: GGGUUUU. In a wheat germ translation system, BYDV-PAV genomic RNA from virions frameshifted about twice as efficiently as full-length transcripts from a cDNA clone. Frameshifting in rabbit reticulocyte lysates was much lower for either template. The identity of the 99-kDa wheat germ translation product was verified as the transframe protein by immunoprecipitation with antibody specific for the 60K ORF. These results support our previous observations of frameshifting in protoplasts and illustrate a subtle molecular control mechanism between this pathogen and its host cells.

Virus-induced gene silencing.

In the postgenomic era, large-scale functional genomic approaches are necessary for converting sequence information into functional information. A para-genetic approach, called virus-induced gene silencing (VIGS), offers a rapid means of gaining insight into gene function in plants. VIGS system could be used to suppress endogenous gene expression by infecting plants with a recombinant virus vector (VIGS vector) carrying host-derived sequence. Here, we describe the use of tobacco rattle virus (TRV)-based VIGS technique to study gene function in Nicotiana benthamiana and tomato.

Extreme Reduction of Disease in Oats Transformed with the 5' Half of the Barley Yellow Dwarf Virus-PAV Genome.

ABSTRACT Barley yellow dwarf viruses (BYDVs) are the most serious and widespread viruses of oats, barley, and wheat worldwide. Natural resistance is inadequate. Toward overcoming this limitation, we engineered virus-derived transgenic resistance in oat. Oat plants were transformed with the 5' half of the BYDV strain PAV genome, which includes the RNA-dependent RNA polymerase gene. In experiments on T2- and T3-generation plants descended from the same transformation event, all BYDV-inoculated plants containing the transgene showed disease symptoms initially, but recovered, flowered, and produced seed. In contrast, all but one of the BYDV-PAV-inoculated nontransgenic segregants died before reaching 25 cm in height. Although all of the recovered transgenic plants looked similar, the amount of virus and viral RNA ranged from substantial to undetectable levels. Thus, the transgene may act either by restricting virus accumulation or by a novel transgenic tolerance phenomenon. This work demonstrates a strategy for genetically stable transgenic resistance to BYDVs that should apply to all hosts of the virus.

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates.

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4's specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.

Studying NB-LRR immune receptor localization by agroinfiltration transient expression.

NB-LRR immune receptors in plants play dual roles as sentries and as activators of defense. The site in the cell where these activities take place can be different for different NB-LRRs. Furthermore, recognition and defense activation can occur in distinct subcellular compartments. Therefore, determining the subcellular localization of NB-LRRs is a key step toward understanding how they function. Recent advances in confocal microscopy enable high-resolution imaging of proteins in live cells. Agroinfiltration in the Nicotiana benthamiana model plant system is a convenient way of expressing proteins for localization studies. This chapter explains how to use N. benthamiana to transiently express NB-LRRs for confocal fluorescence microscopy.

Virus-induced gene silencing in nicotiana benthamiana and other plant species.

Virus-induced gene silencing (VIGS) is an efficient tool for high throughput reverse genetic screens. VIGS engages the endogenous RNA-silencing machinery of the plant host, and can yield an 85-95% reduction of target transcripts. Gene silencing is rapid, target-specific, and does not require the creation of stable transformants. The technique has been used successfully in numerous Solanaceae species as well as in Arabidopsis, maize, and rice. Here we describe a protocol for conducting a VIGS screen in Nicotiana benthamiana using Tobacco Rattle Virus (TRV) based silencing vectors. This protocol can readily be adapted to many other model plant species.

What can plant autophagy do for an innate immune response?

Autophagy plays an established role in the execution of senescence, starvation, and stress responses in plants. More recently, an emerging role for autophagy has been discovered during the plant innate immune response. Recent papers have shown autophagy to restrict, and conversely, to also promote programmed cell death (PCD) at the site of pathogen infection. These initial studies have piqued our excitement, but they have also revealed gaps in our understanding of plant autophagy regulation, in our ability to monitor autophagy in plant cells, and in our ability to manipulate autophagic activity. In this review, we present the most pressing questions now facing the field of plant autophagy in general, with specific focus on autophagy as it occurs during a plant-pathogen interaction. To begin to answer these questions, we place recent findings in the context of studies of autophagy and immunity in other systems, and in the context of the mammalian immune response in particular.

Virus-induced gene silencing offers a functional genomics platform for studying plant cell wall formation.

Virus-induced gene silencing (VIGS) is a powerful genetic tool for rapid assessment of plant gene functions in the post-genomic era. Here, we successfully implemented a Tobacco Rattle Virus (TRV)-based VIGS system to study functions of genes involved in either primary or secondary cell wall formation in Nicotiana benthamiana plants. A 3-week post-VIGS time frame is sufficient to observe phenotypic alterations in the anatomical structure of stems and chemical composition of the primary and secondary cell walls. We used cell wall glycan-directed monoclonal antibodies to demonstrate that alteration of cell wall polymer synthesis during the secondary growth phase of VIGS plants has profound effects on the extractability of components from woody stem cell walls. Therefore, TRV-based VIGS together with cell wall component profiling methods provide a high-throughput gene discovery platform for studying plant cell wall formation from a bioenergy perspective.