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Autophagy plays a paramount role in mammalian antiviral immunity including direct targeting of viruses and their individual components, and many viruses have evolved measures to antagonize or even exploit autophagy mechanisms for the benefit of infection. In plants, however, the functions of autophagy in host immunity and viral pathogenesis are poorly understood. In this study, we have identified both anti- and proviral roles of autophagy in the compatible interaction of cauliflower mosaic virus (CaMV), a double-stranded DNA pararetrovirus, with the model plant Arabidopsis thaliana We show that the autophagy cargo receptor NEIGHBOR OF BRCA1 (NBR1) targets nonassembled and virus particle-forming capsid proteins to mediate their autophagy-dependent degradation, thereby restricting the establishment of CaMV infection. Intriguingly, the CaMV-induced virus factory inclusions seem to protect against autophagic destruction by sequestering capsid proteins and coordinating particle assembly and storage. In addition, we found that virus-triggered autophagy prevents extensive senescence and tissue death of infected plants in a largely NBR1-independent manner. This survival function significantly extends the timespan of virus production, thereby increasing the chances for virus particle acquisition by aphid vectors and CaMV transmission. Together, our results provide evidence for the integration of selective autophagy into plant immunity against viruses and reveal potential viral strategies to evade and adapt autophagic processes for successful pathogenesis.

Cauliflower mosaic virus (CaMV) encodes a multifunctional protein P6 that is required for translation of the 35S RNA and also acts as a suppressor of RNA silencing. Here we demonstrate that P6 additionally acts as a pathogenicity effector of an unique and novel type, modifying NPR1 (a key regulator of salicylic acid (SA)- and jasmonic acid (JA)-dependent signaling) and inhibiting SA-dependent defence responses We find that that transgene-mediated expression of P6 in Arabidopsis and transient expression in Nicotiana benthamiana has profound effects on defence signaling, suppressing expression of representative SA-responsive genes and increasing expression of representative JA-responsive genes. Relative to wild-type Arabidopsis P6-expressing transgenics had greatly reduced expression of PR-1 following SA-treatment, infection by CaMV or inoculation with an avirulent bacterial pathogen Pseudomonas syringae pv tomato (Pst). Similarly transient expression in Nicotiana benthamiana of P6 (including a mutant form defective in translational transactivation activity) suppressed PR-1a transcript accumulation in response to Agrobacterium infiltration and following SA-treatment. As well as suppressing the expression of representative SA-regulated genes, P6-transgenic Arabidopsis showed greatly enhanced susceptibility to both virulent and avirulent Pst (titres elevated 10 to 30-fold compared to non-transgenic controls) but reduced susceptibility to the necrotrophic fungus Botrytis cinerea. Necrosis following SA-treatment or inoculation with avirulent Pst was reduced and delayed in P6-transgenics. NPR1 an important regulator of SA/JA crosstalk, was more highly expressed in the presence of P6 and introduction of the P6 transgene into a transgenic line expressing an NPR1:GFP fusion resulted in greatly increased fluorescence in nuclei even in the absence of SA. Thus in the presence of P6 an inactive form of NPR1 is mislocalized in the nucleus even in uninduced plants. These results demonstrate that P6 is a new type of pathogenicity effector protein that enhances susceptibility to biotrophic pathogens by suppressing SA- but enhancing JA-signaling responses.

Optical spectroscopy can be used to quickly characterise the structural properties of individual molecules. However, it cannot be applied to biological assemblies because light is generally blind to the spatial distribution of the component molecules. This insensitivity arises from the mismatch in length scales between the assemblies (a few tens of nm) and the wavelength of light required to excite chromophores (≥150 nm). Consequently, with conventional spectroscopy, ordered assemblies, such as the icosahedral capsids of viruses, appear to be indistinguishable isotropic spherical objects. This limits potential routes to rapid high-throughput portable detection appropriate for point-of-care diagnostics. Here, we demonstrate that chiral electromagnetic (EM) near fields, which have both enhanced chiral asymmetry (referred to as superchirality) and subwavelength spatial localisation (∼10 nm), can detect the icosahedral structure of virus capsids. Thus, they can detect both the presence and relative orientation of a bound virus capsid. To illustrate the potential uses of the exquisite structural sensitivity of subwavelength superchiral fields, we have used them to successfully detect virus particles in the complex milieu of blood serum.Bioimaging: Spotting viral signatures with ‘superchiral’ lightA technique that uses twisted light fields to detect biomolecular structures could find application as a low-cost clinical tool for screening viruses. The protein coatings around many viruses, such as the turnip yellow mosaic virus (TYMV), have complex polyhedral shapes that are difficult to resolve with conventional optical microscopes. Malcolm Kadodwala from the University of Glasgow and other colleagues in the United Kingdom now report that ‘superchiral’ light — localized fields generated by metal nanostructures that spiral as they travel — are sensitive to the asymmetric polyhedral of TYMV. By spectroscopic measurements of particle rotations in superchiral light at different frequencies, the team identified specific asymmetric signals that correlated to virus alignment on gold photonic substrates. This approach was then used to determine TYMV levels in human blood serum spiked with the virus.

Summary The plant pathogen, Pseudomonas syringae (Ps), together with related Ps species, infects and attacks a wide range of agronomically important crops, including tomato, kiwifruit, pepper, olive and soybean, causing economic losses. Currently, chemicals and introduced resistance genes are used to protect plants against these pathogens but have limited success and may have adverse environmental impacts. Consequently, there is a pressing need to develop alternative strategies to combat bacterial disease in crops. One such strategy involves using narrow‐spectrum protein antibiotics (so‐called bacteriocins), which diverse bacteria use to compete against closely related species. Here, we demonstrate that one bacteriocin, putidacin L1 (PL1), can be expressed in an active form at high levels in Arabidopsis and in Nicotiana benthamiana in planta to provide effective resistance against diverse pathovars of Ps. Furthermore, we find that Ps strains that mutate to acquire tolerance to PL1 lose their O‐antigen, exhibit reduced motility and still cannot induce disease symptoms in PL1‐transgenic Arabidopsis. Our results provide proof‐of‐principle that the transgene‐mediated expression of a bacteriocin in planta can provide effective disease resistance to bacterial pathogens. Thus, the expression of bacteriocins in crops might offer an effective strategy for managing bacterial disease, in the same way that the genetic modification of crops to express insecticidal proteins has proven to be an extremely successful strategy for pest management. Crucially, nearly all genera of bacteria, including many plant pathogenic species, produce bacteriocins, providing an extensive source of these antimicrobial agents.

Iron is a limiting nutrient in bacterial infection putting it at the centre of an evolutionary arms race between host and pathogen. Gram-negative bacteria utilize TonB-dependent outer membrane receptors to obtain iron during infection. These receptors acquire iron either in concert with soluble iron-scavenging siderophores or through direct interaction and extraction from host proteins. Characterization of these receptors provides invaluable insight into pathogenesis. However, only a subset of virulence-related TonB-dependent receptors have been currently described. Here we report the discovery of FusA, a new class of TonB-dependent receptor, which is utilized by phytopathogenic Pectobacterium spp. to obtain iron from plant ferredoxin. Through the crystal structure of FusA we show that binding of ferredoxin occurs through specialized extracellular loops that form extensive interactions with ferredoxin. The function of FusA and the presence of homologues in clinically important pathogens suggests that small iron-containing proteins represent an iron source for bacterial pathogens. Many bacteria use TonB-dependent outer membrane receptors to scavenge iron from their host during infection. Here, the authors report on the structure and function of FusA, which is a bacterial receptor that is used to obtain iron from plants.

We analyzed expression of marker genes for three defense pathways during infection by Cauliflower mosaic virus (CaMV), a compatible pathogen of Arabidopsis (Arabidopsis thaliana), using luciferase reporter transgenes and directly by measuring transcript abundance. Expression of PR-1, a marker for salicylic acid signaling, was very low until 8 d postinoculation and then rose sharply, coinciding with the rise in virus levels. In contrast, as early as 2 h postinoculation, transcriptional up-regulation of GST1--a marker for reactive oxygen species--and PDF1.2--a marker for jasmonic acid/ethylene defense signaling--was detectable in the virus-inoculated leaf and systemically. In parallel with the activation of GST1, H₂O₂ accumulated locally and systemically in virus- but not mock-inoculated plants. However, in plants inoculated with infectious CaMV DNA rather than virus particles, the onset of systemic luciferase activity was delayed by 24 to 48 h, suggesting that virion structural proteins act as the elicitor. This phenomenon, which we term the rapid systemic response, preceded virus movement from the inoculated leaf; therefore, the systemic signal is not viral. Systemic, but not local, H₂O₂ accumulation was abolished in rbohDF double mutants and in etr1-1 and ein2-1 mutants, implicating NADPH oxidase and ethylene signaling in the generation and transduction of the response. Ethylene, but not rbohDF mutants, also showed reduced susceptibility to CaMV, whereas in NahG transgenics, virus levels were similar to wild type. These findings implicate reactive oxygen species and ethylene in signaling in response to CaMV infection, but suggest that salicylic acid does not play an effective role.

Long-distance virus transport takes place through the vascular system and is dependent on the movement of photoassimilates. Here, patterns of symptom development, virus movement and gene expression were analysed in Arabidopsis following inoculation with Cauliflower mosaic virus (CaMV) on a single leaf. Virus accumulation and expression of markers for the salicylic acid (SA) and ethylene/jasmonate (Et/JA) defence pathways, PR-1 and PDF1.2, were analysed on a leaf-by-leaf basis by real-time reverse transcription polymerase chain reaction (qRT-PCR). Virus spread followed a strictly defined pattern identical to that of a source-sink relationship. This was exploited to study differences between local and systemic defence responses in a developmental and spatial manner. In infected plants, PR-1 transcripts accumulated primarily but not exclusively in leaves with a direct vascular connection to the inoculated leaf. Abundances fell significantly as virus accumulated. By contrast, PDF1.2 transcripts were significantly lower than in controls in all leaves at early stages of infection, but recovered as virus accumulated. Virus and PR-1 transcript abundances are negatively correlated, and SA- and Et/JA-mediated signalling of gene expression occurs independently of the presence of virus. Although SA-dependent signalling responses were mainly linked to the orthostichy, Et/JA-dependent responses were independent of vascular connections.

PR-1 has been extensively used as a marker for salicylic acid (SA)-mediated defence and systemic and local acquired resistance. The Arabidopsis Genome Project annotates At2g19990 as PR-1. This gene is also identified as PR-1 in two \"full genome\" Arabidopsis microarrays, and TAIR cites approximately 60 articles to describe its patterns of expression. However, most of these citations are incorrect; the probes used were not At2g19990, but a homologous gene At2g14610, which is annotated as \"PR-1-like\". Because of the potential for confusion, we analyzed the expression of both genes in Arabidopsis thaliana (L.) Heynh. At2g14610 (PR-1-like) showed the archetypal patterns of SA-responsive expression: mRNA levels increased following SA-treatment, inoculation with an avirulent (but not a virulent) strain of Pseudomonas syringae, and in wild-type (but not NahG) Arabidopsis infected with cauliflower mosaic virus (CaMV). In cpr5 mutants it was expressed constitutively. In contrast, expression of At2g19990 (annotated as PR-1) was detectable in neither SA-treated Col-0 nor in cpr5. Infection by virulent and avirulent isolates of P. syringae up-regulated expression, but to a similar level, and infection by CaMV induced a modest increase in expression in both the wild type and NahG. At2g19990, although pathogen responsive, does not show the SA-dependent patterns of expression expected from a member of the PR-1 regulon, and its annotation as \"PR-1\" is inappropriate. The annotations should identify At2g14610 as the authentic PR-1.

Protein P6 is the main symptom determinant of cauliflower mosaic virus (CaMV), and transgene-mediated expression in Arabidopsis induces a symptom-like phenotype in the absence of infection. Seeds of a P6-transgenic line, A7, were mutagenized by gamma-irradiation and M2 seedlings were screened for mutants that suppressed the phenotype of chlorosis and stunting. We identified four mutants that were larger and less chlorotic than the A7 parent but which contained an intact and transcriptionally active transgene. The two mutants with the strongest suppression phenotype, were recessive and allelic. The transgene was eliminated by back-crossing with wild-type Arabidopsis. In progeny lines that were homozygous for the putative suppressor mutation the proportion of plants becoming infected following inoculation with CaMV was 40% that of wild-type, although in plants that did become infected, levels of virus DNA in mutants and wild-type did not differ significantly. Symptoms in the mutants were milder and delayed although this was somewhat dependent on the virus isolate. This phenotype was inherited stably. Both mutant alleles showed a partially ethylene-insensitive phenotype in an ethylene triple response assay. P6-transgenic plants were also almost completely insensitive to ethylene in the triple response assay. We suggest that the chlorosis and stunting in P6-transgenic and CaMV-infected plants are dependent on interactions between P6 and components involved in ethylene signalling, and that the suppressor gene product may function to augment these interactions.

The contents of single plant cells can be sampled using glass microcapillaries. By combining such single-cell sampling with reverse transcription-polymerase chain reaction (RT-PCR), transcripts of individual genes can be identified and, in principle, quantified. This provides a valuable technique for the analysis and quantification of the intercellular distribution of gene expression in complex tissues. In a proof-of-principle study, the cellular locations of the transcripts of the eight isoforms of actin (ACT) expressed in Arabidopsis thaliana (L.) Heynh. were analyzed. Cell sap was extracted from epidermal and mesophyll cells of leaves of 3- to 4-week-old plants. Single-cell (SC)-RT-PCR was used to amplify the actin transcripts using specific primer pairs for ACT1, 2, 3, 4, 7, 8, 11 and 12. Only ACT2 and ACT8 were found in epidermal and in mesophyll cells. In individual trichomes, in addition to ACT2 and ACT8, ACT7 and ACT11 transcripts were detectable. By employing the already well-characterized actin system we demonstrate the practicality and power of SC-RT-PCR as a technique for analyzing gene expression at the ultimate level of resolution, the single cell.