MiR827 positively regulates the resistance to chilli veinal mottle virus by affecting the expression of FBPase in Nicotiana benthamiana.

MicroRNA(miRNA) is a class of non-coding small RNA that plays an important role in plant growth, development, and response to environmental stresses. Unlike most miRNAs, which usually target homologous genes across a variety of species, miR827 targets different types of genes in different species. Research on miR827 mainly focuses on its role in regulating phosphate (Pi) homeostasis of plants, however, little is known about its function in plant response to virus infection. In the present study, miR827 was significantly upregulated in the recovery tissue of virus-infected Nicotiana tabacum. Overexpression of miR827 could improve plants resistance to the infection of chilli veinal mottle virus (ChiVMV) in Nicotiana benthamiana, whereas interference of miR827 increased the susceptibility of the virus-infected plants. Further experiments indicated that the antiviral defence regulated by miR827 was associated with the reactive oxygen species and salicylic acid signalling pathways. Then, fructose-1,6-bisphosphatase (FBPase) was identified to be a target of miR827, and virus infection could affect the expression of FBPase. Finally, transient expression of FBPase increased the susceptibility to ChiVMV-GFP infection in N. benthamiana. By contrast, silencing of FBPase increased plant resistance. Taken together, our results demonstrate that miR827 plays a positive role in tobacco response to virus infection, thus providing new insights into understanding the role of miR827 in plant-virus interaction.

An Agent-Based Model Shows How Mixed Infections Drive Multiyear Pathotype Dynamics in a Plant-Virus System.

Mathematical models are widely used to understand the evolution and epidemiology of plant pathogens under a variety of scenarios. Here, we used this approach to analyze the effects of different traits intrinsic and extrinsic to plant-virus interactions on the dynamics of virus pathotypes in genetically heterogeneous plant-virus systems. For this, we propose an agent-based epidemiological model that includes epidemiologically significant pathogen life-history traits related to virulence, transmission, and survival in the environment and allows for integrating long- and short-distance transmission, primary and secondary infections, and within-host pathogen competition in mixed infections. The study focuses on the tobamovirus-pepper pathosystem. Model simulations allowed us to integrate pleiotropic effects of resistance-breaking mutations on different virus life-history traits into the net costs of resistance breaking, allowing for predictions on multiyear pathotype dynamics. We also explored the effects of two control measures, the use of host resistance and roguing of symptomatic plants, that modify epidemiological attributes of the pathogens to understand how their populations will respond to evolutionary pressures. One major conclusion points to the importance of pathogen competition within mixed-infected hosts as a component of the overall fitness of each pathogen that, thus, drives their multiyear dynamics.

iTRAQ-Based Proteomic Analysis of Watermelon Fruits in Response to Cucumber green mottle mosaic virus Infection.

Cucumber green mottle mosaic virus (CGMMV) is an important viral pathogen on cucurbit plants worldwide, which can cause severe fruit decay symptoms on infected watermelon (usually called "watermelon blood flesh"). However, the molecular mechanism of this disease has not been well understood. In this study, we employed the isobaric tags for relative and absolute quantitation (iTRAQ) technique to analyze the proteomic profiles of watermelon fruits in response to CGMMV infection. A total of 595 differentially accumulated proteins (DAPs) were identified, of which 404 were upregulated and 191 were downregulated. Functional annotation analysis showed that these DAPs were mainly involved in photosynthesis, carbohydrate metabolism, secondary metabolite biosynthesis, plant-pathogen interaction, and protein synthesis and turnover. The accumulation levels of several proteins related to chlorophyll metabolism, pyruvate metabolism, TCA cycle, heat shock proteins, thioredoxins, ribosomal proteins, translation initiation factors, and elongation factors were strongly affected by CGMMV infection. Furthermore, a correlation analysis was performed between CGMMV-responsive proteome and transcriptome data of watermelon fruits obtained in our previous study, which could contribute to comprehensively elucidating the molecular mechanism of "watermelon blood flesh". To confirm the iTRAQ-based proteome data, the corresponding transcripts of ten DAPs were validated by determining their abundance via quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). These results could provide a scientific basis for in-depth understanding of the pathogenic mechanisms underlying CGMMV-induced "watermelon blood flesh", and lay the foundation for further functional exploration and verification of related genes and proteins.

Artificial microRNA-mediated resistance to cucumber green mottle mosaic virus in Nicotiana benthamiana.

MAIN CONCLUSION: We describe a Nicotiana benthamiana system for rapid identification of artificial microRNA (amiRNA) to control cucumber green mottle mosaic virus (CGMMV) disease. Although artificial miRNA technology has been used to control other viral diseases, it has not been applied to reduce severe cucumber green mottle mosaic virus (CGMMV) disease and crop loss in the economically important cucurbits. We used our system to identify three amiRNAs targeting CGMMV RNA (amiR1-CP, amiR4-MP and amiR6-Rep) and show that their expression reduces CGMMV replication and disease in virus-infected plants. This work streamlines the process of generating amiRNA virus-resistant crops and can be broadly applied to identify active antiviral amiRNAs against a broad spectrum of viruses to control disease in diverse crops.

Expression profiling and regulatory network of cucumber microRNAs and their putative target genes in response to cucumber green mottle mosaic virus infection.

Cucumber green mottle mosaic virus (CGMMV) is an important pathogen of cucumber (Cucumis sativus). The molecular mechanisms mediating host-pathogen interactions are likely to be strongly influenced by microRNAs (miRNAs), which are known to regulate gene expression during the disease cycle. This study focused on 14 miRNAs (miR159, miR169, miR172, miR838, miR854, miR5658, csa-miRn1-3p, csa-miRn2-3p, csa-miRn3-3p, csa-miRn4-5p, csa-miRn5-5p, csa-miRn6-3p, csa-miRn7-5p and csa-miRn8-3p) and their target genes. The data collected was used to construct a regulatory network of miRNAs and target genes associated with cucumber-CGMMV interactions, which identified 608 potential target genes associated with all of the miRNAs except csa-miRn7-5p. Five of the miRNAs (miR159, miR838, miR854, miR5658 and csa-miRn6-3p) were found to be mutually linked by target genes, while another eight (miR169, miR172, csa-miRn1-3p, csa-miRn2-3p, csa-miRn3-3p, csa-miRn4-5p, csa-miRn5-5p and csa-miRn8-3p) formed subnetworks that did not display any connectivity with other miRNAs or their target genes. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to analyze the expression levels of the different miRNAs and their putative target genes in leaf, stem and root samples of cucumber over a 42-day period after inoculation with CGMMV. A positive correlation was found between some of the miRNAs and their respective target genes, although for most, the response varied greatly depending on the time point, indicating that additional factors are likely to be involved in the interaction between cucumber miRNAs and their target genes. Several miRNAs, including miR159 and csa-miRn6-3p, were linked to target genes that have been associated with plant responses to disease. A model linking miRNAs, their targets and downstream biological processes is proposed to indicate the roles of these miRNAs in the cucumber-CGMMV pathosystem.

Development of a new tobamovirus-based viral vector for protein expression in plants.

Plants are becoming an interesting alternative system for the heterologous production of pharmaceutical proteins, providing a more scalable, cost-effective, and biologically safer option than the current expression systems. The development of plant virus expression vectors has allowed rapid and high-level transient expression of recombinant genes, and, in turn, provided an attractive plant-based production platform. Here we report the development of vectors based on the tobamovirus Pepper mild mottle virus (PMMoV) to be used in transient expression of foreign genes. In this PMMoV vector, a middle part of the viral coat protein gene was replaced by the green fluorescent protein (GFP) gene, and this recombinant genome was assembled in a binary vector suitable for plant agroinoculation. The accumulation of GFP was evaluated by observation of green fluorescent signals under UV light and by western blotting. Furthermore, by using this vector, the multiepitope gene for chikungunya virus was successfully expressed and confirmed by western blotting. This PMMoV-based vector represents an alternative system for a high-level production of heterologous protein in plants.

Understanding the Transmissibility of Cucumber Green Mottle Mosaic Virus in Watermelon Seeds and Seed Health Assays.

Cucumber green mottle mosaic virus (CGMMV), an emerging tobamovirus, has caused serious disease outbreaks to cucurbit crops in several countries, including the United States. Although CGMMV is seed-borne, the mechanism of its transmission from a contaminated seed to germinating seedling is still not fully understood, and the most suitable seed health assay method has not been well established. To evaluate the mechanism of seed transmissibility, using highly contaminated watermelon seeds collected from CGMMV-infected experimental plants, bioassays were conducted in a greenhouse through seedling grow-out and by mechanical inoculation. Through natural seedling grow-out, we did not observe seed transmission of CGMMV to germinating seedlings. However, efficient transmission of CGMMV was observed using bioassays on melon plants through mechanical inoculation of seed extract prepared from CGMMV-contaminated seeds. Understanding the seed-borne property and the ease of mechanical transmission of CGMMV from a contaminated seed to seedling is an important finding. In comparative evaluation of various laboratory techniques for seed health assays, we found that enzyme-linked immunosorbent assay and loop-mediated isothermal amplification were the most sensitive and reliable methods to detect CGMMV on cucurbit seeds. Because CGMMV is a seed-borne and highly contagious virus, a new infection might not result in a natural seedling grow-out; it could occur through mechanical transmission from contaminated seeds. Therefore, a sensitive seed health test is necessary to ensure CGMMV-free seed lots are used for planting.

Insight Into Late Wilting Disease of Cucumber Demonstrates the Complexity of the Phenomenon in Fluctuating Environments.

Some diseases are caused by coinfection of several pathogens in the same plant. However, studies on the complexity of these coinfection events under different environmental conditions are scarce. Our ongoing research involves late wilting disease of cucumber caused by coinfection of Cucumber green mottle mosaic virus (CGMMV) and Pythium spp. We specifically investigated the role of various temperatures (18, 25, 32°C) on the coinfection by CGMMV and two predominant Pythium species occurring in cucumber greenhouses under Middle Eastern climatic conditions. During the summer months, Pythium aphanidermatum was most common, whereas P. spinosum predominated during the winter-spring period. P. aphanidermatum preferred higher temperatures while P. spinosum preferred low temperatures and caused very low levels of disease at 32°C when the 6-day-old seedlings were infected with P. spinosum alone. Nevertheless, after applying a later coinfection with CGMMV on the 14-day-old plants, a synergistic effect was detected for both Pythium species at optimal and suboptimal temperatures, with P. spinosum causing high mortality incidence even at 32°C. The symptoms caused by CGMMV infection appeared earlier as the temperature increased. However, within each temperature, no significant influence of the combined infection was detected. Our results demonstrate the complexity of coinfection in changing environmental conditions and indicate its involvement in disease development and severity as compared with infection by each of the pathogens alone.

Revisiting the Roles of Tobamovirus Replicase Complex Proteins in Viral Replication and Silencing Suppression.

Tobamoviral replicase possesses an RNA-dependent RNA polymerase (RDR) domain and is translated from genomic (g)RNA via a stop codon readthrough mechanism at a one-to-ten ratio relative to a shorter protein lacking the RDR domain. The two proteins share methyltransferase and helicase domains and form a heterodimer implicated in gRNA replication. The shorter protein is also implicated in suppressing RNA silencing-based antiviral defenses. Using a stop codon mutant of Oilseed rape mosaic tobamovirus (ORMV), we demonstrate that the readthrough replicase (p182) is sufficient for gRNA replication and for subgenomic RNA transcription during systemic infection in Nicotiana benthamiana and Arabidopsis thaliana. However, the mutant virus displays milder symptoms and does not interfere with HEN1-mediated methylation of viral short interfering (si)RNAs or plant small (s)RNAs. The mutant virus tends to revert the stop codon, thereby restoring expression of the shorter protein (p125), even in the absence of plant Dicer-like activities that generate viral siRNAs. Plant RDR activities that generate endogenous siRNA precursors do not prevent replication or movement of the mutant virus, and double-stranded precursors of viral siRNAs representing the entire virus genome are likely synthesized by p182. Transgenic expression of p125 partially recapitulates the ORMV disease symptoms associated with overaccumulation of plant sRNAs. Taken together, the readthrough replicase p182 is sufficient for viral replication and transcription but not for silencing suppression. By contrast, the shorter p125 protein suppresses silencing, provokes severe disease symptoms, causes overaccumulation of unmethylated viral and plant sRNAs but it is not an essential component of the viral replicase complex.

Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses.

Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant-virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses.

Conferring virus resistance in tomato by independent RNA silencing of three tomato homologs of Arabidopsis TOM1.

The TOM1/TOM3 genes from Arabidopsis are involved in the replication of tobamoviruses. Tomato homologs of these genes, LeTH1, LeTH2 and LeTH3, are known. In this study, we examined transgenic tomato lines where inverted repeats of either LeTH1, LeTH2 or LeTH3 were introduced by Agrobacterium. Endogenous mRNA expression for each gene was detected in non-transgenic control plants, whereas a very low level of each of the three genes was found in the corresponding line. Small interfering RNA was detected in the transgenic lines. Each silenced line showed similar levels of tobamovirus resistance, indicating that each gene is similarly involved in virus replication.

Non-structural Functions of Hordeivirus Capsid Protein Identified in Plants Infected by a Chimeric Tobamovirus.

Capsid proteins (CPs) of (+)RNA-containing plant viruses are multifunctional proteins involved in many stages of viral infection cycle, in addition to their main function of virus capsid formation. For example, the tobamoviral CP ensures virus systemic transport in plants and defines the virus-host interactions, thereby influencing the virus host range, virus infectivity, pathogenicity, and manifestation of infection symptoms. Hordeiviruses and tobamoviruses belong to the Virgaviridae family and have rod-shaped virions with a helical symmetry; their CPs are similar in structure. However, no non-structural functions of hordeiviral CPs have been described so far. In this study, we assayed possible non-structural functions of CP from the barley stripe mosaic virus (BSMV) (hordeivirus). To do this, the genome of turnip vein clearing virus (TVCV) (tobamovirus) was modified by substituting the TVCV CP gene with the BSMV CP gene or its mutants. We found that BSMV CP efficiently replaced TVCV CP at all stages of viral infection. In particular, BSMV CP performed the role of tobamoviral CP in the long-distance transport of the chimeric virus, acted as a hypersensitive response elicitor, and served as a pathogenicity determinant that influenced the symptoms of the viral infection. The chimeric tobamovirus coding for the C-terminally truncated BSMV CP displayed an increased infectivity and was transported in plants in a form of atypical virions (ribonucleoprotein complexes).

Combined Infection with Cucumber green mottle mosaic virus and Pythium Species Causes Extensive Collapse in Cucumber Plants.

In the last decade, the phenomenon of late-wilting has increased in cucumber greenhouses during Cucumber green mottle mosaic virus (CGMMV) epidemics. Because the wilting appears in defined patches accompanied by root rot, it was hypothesized that the phenomenon is caused by coinfection of soilborne pathogens and CGMMV. A field survey showed that 69% of the collapsed plants were infected with both Pythium spp. and CGMMV, whereas only 20 and 6.6% were singly infected with Pythium spp. or CGMMV, respectively. Artificial inoculations in controlled-environmental growth chambers and glasshouse experiments showed that coinfection with Pythium spinosum and CGMMV leads to a strong synergistic wilting effect and reduces growth parameters. The synergy values of the wilting effect were not influenced by the time interval between P. spinosum and CGMMV infection. However, dry mass synergy values were decreased with longer intervals between infections. The results obtained in this study support the complexity of the wilting phenomenon described in commercial cucumber grown in protected structures during infection of Pythium spp. on the background of a vast CGMMV epidemic. They encourage a wider perspective of the complexity of agricultural diseases to apply the most suitable disease management.

Biological and Genomic Characterization of a Novel Tobamovirus Infecting Hoya spp.

Foliar symptoms suggestive of virus infection were observed on the ornamental plant hoya (Hoya spp.; commonly known as waxflower) in Florida. An agent that reacted with commercially available tobamovirus detection reagents was mechanically transmitted to Chenopodium quinoa and Nicotiana benthamiana. Rod-shaped particles ∼300 nm in length and typical of tobamoviruses were observed in partially purified virion preparations by electron microscopy. An experimental host range was determined by mechanical inoculation with virions, and systemic infections were observed in plants in the Asclepiadaceae, Apocynaceae, and Solanaceae families. Some species in the Solanaceae and Chenopodiaceae families allowed virus replication only in inoculated leaves, and were thus only local hosts for the virus. Tested plants in the Amaranthaceae, Apiaceae, Brassicaceae, Cucurbitaceae, Fabaceae, and Malvaceae did not support either local or systemic virus infection. The complete genome for the virus was sequenced and shown to have a typical tobamovirus organization. Comparisons of genome nucleotide sequence and individual gene deduced amino acid sequences indicate that it is a novel tobamovirus sharing the highest level of sequence identity with Streptocarpus flower break virus and members of the Brassicaceae-infecting subgroup of tobamoviruses. The virus, for which the name Hoya chlorotic spot virus (HoCSV) is proposed, was detected in multiple hoya plants from different locations in Florida.

Mutations That Determine Resistance Breaking in a Plant RNA Virus Have Pleiotropic Effects on Its Fitness That Depend on the Host Environment and on the Type, Single or Mixed, of Infection.

UNLABELLED: Overcoming host resistance in gene-for-gene host-virus interactions is an important instance of host range expansion, which can be hindered by across-host fitness trade-offs. Trade-offs are generated by negative effects of host range mutations on the virus fitness in the original host, i.e., by antagonistic pleiotropy. It has been reported that different mutations in Pepper mild mottle virus (PMMoV) coat protein result in overcoming L-gene resistance in pepper. To analyze if resistance-breaking mutations in PMMoV result in antagonistic pleiotropy, all reported mutations determining the overcoming of L(3) and L(4) alleles were introduced in biologically active cDNA clones. Then, the parental and mutant virus genotypes were assayed in susceptible pepper genotypes with an L(+), L(1), or L(2) allele, in single and in mixed infections. Resistance-breaking mutations had pleiotropic effects on the virus fitness that, according to the specific mutation, the host genotype, and the type of infection, single or mixed with other virus genotypes, were antagonistic or positive. Thus, resistance-breaking mutations can generate fitness trade-offs both across hosts and across types of infection, and the frequency of host range mutants will depend on the genetic structure of the host population and on the frequency of mixed infections by different virus genotypes. Also, resistance-breaking mutations variously affected virulence, which may further influence the evolution of host range expansion. IMPORTANCE: A major cause of virus emergence is host range expansion, which may be hindered by across-host fitness trade-offs caused by negative pleiotropy of host range mutations. An important instance of host range expansion is overcoming host resistance in gene-for-gene plant-virus interactions. We analyze here if mutations in the coat protein of Pepper mild mottle virus determining L-gene resistance-breaking in pepper have associated fitness penalties in susceptible host genotypes. Results show that pleiotropic effects of resistance-breaking mutations on virus fitness depend on the specific mutation, the susceptible host genotype, and the type of infection, single or mixed, with other virus genotypes. Accordingly, resistance-breaking mutations can have negative, positive, or no pleiotropic effects on virus fitness. These results underscore the complexity of host range expansion evolution and, specifically, the difficulty of predicting the overcoming of resistance factors in crops.

The complete genome sequence, occurrence and host range of Tomato mottle mosaic virus Chinese isolate.

BACKGROUND: Tomato mottle mosaic virus (ToMMV) is a recently identified species in the genus Tobamovirus and was first reported from a greenhouse tomato sample collected in Mexico in 2013. In August 2013, ToMMV was detected on peppers (Capsicum spp.) in China. However, little is known about the molecular and biological characteristics of ToMMV. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and rapid identification of cDNA ends (RACE) were carried out to obtain the complete genomic sequences of ToMMV. Sap transmission was used to test the host range and pathogenicity of ToMMV. RESULTS: The full-length genomes of two ToMMV isolates infecting peppers in Yunnan Province and Tibet Autonomous Region of China were determined and analyzed. The complete genomic sequences of both ToMMV isolates consisted of 6399 nucleotides and contained four open reading frames (ORFs) encoding 126, 183, 30 and 18 kDa proteins from the 5' to 3' end, respectively. Overall similarities of the ToMMV genome sequence to those of the other tobamoviruses available in GenBank ranged from 49.6% to 84.3%. Phylogenetic analyses of the sequences of full-genome nucleotide and the amino acids of its four proteins confirmed that ToMMV was most closely related to Tomato mosaic virus (ToMV). According to the genetic structure, host of origin and phylogenetic relationships, the available 32 tobamoviruses could be divided into at least eight subgroups based on the host plant family they infect: Solanaceae-, Brassicaceae-, Cactaceae-, Apocynaceae-, Cucurbitaceae-, Malvaceae-, Leguminosae-, and Passifloraceae-infecting subgroups. The detection of ToMMV on some solanaceous, cucurbitaceous, brassicaceous and leguminous plants in Yunnan Province and other few parts of China revealed ToMMV only occurred on peppers so far. However, the host range test results showed ToMMV could infect most of the tested solanaceous and cruciferous plants, and had a high affinity for the solanaceous plants. CONCLUSIONS: The complete nucleotide sequences of two Chinese ToMMV isolates from naturally infected peppers were verified. The tobamoviruses were divided into at least eight subgroups, with ToMMV belonging to the subgroup that infected plants in the Solanaceae. In China, ToMMV only occurred on peppers in the fields till now. ToMMV could infect the plants in family Solanaceae and Cucurbitaceae by sap transmission.

Modulation of host plant immunity by Tobamovirus proteins.

BACKGROUND: To establish successful infection, plant viruses produce profound alterations of host physiology, disturbing unrelated endogenous processes and contributing to the development of disease. In tobamoviruses, emerging evidence suggests that viral-encoded proteins display a great variety of functions beyond the canonical roles required for virus structure and replication. Among these, their modulation of host immunity appears to be relevant in infection progression. SCOPE: In this review, some recently described effects on host plant physiology of Tobacco mosaic virus (TMV)-encoded proteins, namely replicase, movement protein (MP) and coat protein (CP), are summarized. The discussion is focused on the effects of each viral component on the modulation of host defense responses, through mechanisms involving hormonal imbalance, innate immunity modulation and antiviral RNA silencing. These effects are described taking into consideration the differential spatial distribution and temporality of viral proteins during the dynamic process of replication and spread of the virus. CONCLUSION: In discussion of these mechanisms, it is shown that both individual and combined effects of viral-encoded proteins contribute to the development of the pathogenesis process, with the host plant's ability to control infection to some extent potentially advantageous to the invading virus.

Picturing pathogen infection in plants.

Several imaging techniques have provided valuable tools to evaluate the impact of biotic stress on host plants. The use of these techniques enables the study of plant-pathogen interactions by analysing the spatial and temporal heterogeneity of foliar metabolism during pathogenesis. In this work we review the use of imaging techniques based on chlorophyll fluorescence, multicolour fluorescence and thermography for the study of virus, bacteria and fungi-infected plants. These studies have revealed the impact of pathogen challenge on photosynthetic performance, secondary metabolism, as well as leaf transpiration as a promising tool for field and greenhouse management of diseases. Images of standard chlorophyll fluorescence (Chl-F) parameters obtained during Chl-F induction kinetics related to photochemical processes and those involved in energy dissipation, could be good stress indicators to monitor pathogenesis. Changes on UV-induced blue (F440) and green fluorescence (F520) measured by multicolour fluorescence imaging in pathogen-challenged plants seem to be related with the up-regulation of the plant secondary metabolism and with an increase in phenolic compounds involved in plant defence, such as scopoletin, chlorogenic or ferulic acids. Thermal imaging visualizes the leaf transpiration map during pathogenesis and emphasizes the key role of stomata on innate plant immunity. Using several imaging techniques in parallel could allow obtaining disease signatures for a specific pathogen. These techniques have also turned out to be very useful for presymptomatic pathogen detection, and powerful non-destructive tools for precision agriculture. Their applicability at lab-scale, in the field by remote sensing, and in high-throughput plant phenotyping, makes them particularly useful. Thermal sensors are widely used in crop fields to detect early changes in leaf transpiration induced by both air-borne and soil-borne pathogens. The limitations of measuring photosynthesis by Chl-F at the canopy level are being solved, while the use of multispectral fluorescence imaging is very challenging due to the type of light excitation that is used.

Host resistance selects for traits unrelated to resistance-breaking that affect fitness in a plant virus.

The acquisition by parasites of the capacity to infect resistant host genotypes, that is, resistance-breaking, is predicted to be hindered by across-host fitness trade-offs. All analyses of costs of resistance-breaking in plant viruses have focused on within-host multiplication without considering other fitness components, which may limit understanding of virus evolution. We have reported that host range expansion of tobamoviruses on L-gene resistant pepper genotypes was associated with severe within-host multiplication penalties. Here, we analyze whether resistance-breaking costs might affect virus survival in the environment by comparing tobamovirus pathotypes differing in infectivity on L-gene resistance alleles. We predicted particle stability from structural models, analyzed particle stability in vitro, and quantified virus accumulation in different plant organs and virus survival in the soil. Survival in the soil differed among tobamovirus pathotypes and depended on differential stability of virus particles. Structure model analyses showed that amino acid changes in the virus coat protein (CP) responsible for resistance-breaking affected the strength of the axial interactions among CP subunits in the rod-shaped particle, thus determining its stability and survival. Pathotypes ranked differently for particle stability/survival and for within-host accumulation. Resistance-breaking costs in survival add to, or subtract from, costs in multiplication according to pathotype. Hence, differential pathotype survival should be considered along with differential multiplication to understand the evolution of the virus populations. Results also show that plant resistance, in addition to selecting for resistance-breaking and for decreased multiplication, also selects for changes in survival, a trait unrelated to the host-pathogen interaction that may condition host range expansion.

Coevolution and hierarchical interactions of Tomato mosaic virus and the resistance gene Tm-1.

During antagonistic coevolution between viruses and their hosts, viruses have a major advantage by evolving more rapidly. Nevertheless, viruses and their hosts coexist and have coevolved, although the processes remain largely unknown. We previously identified Tm-1 that confers resistance to Tomato mosaic virus (ToMV), and revealed that it encodes a protein that binds ToMV replication proteins and inhibits RNA replication. Tm-1 was introgressed from a wild tomato species Solanum habrochaites into the cultivated tomato species Solanum lycopersicum. In this study, we analyzed Tm-1 alleles in S. habrochaites. Although most part of this gene was under purifying selection, a cluster of nonsynonymous substitutions in a small region important for inhibitory activity was identified, suggesting that the region is under positive selection. We then examined the resistance of S. habrochaites plants to ToMV. Approximately 60% of 149 individuals from 24 accessions were resistant to ToMV, while the others accumulated detectable levels of coat protein after inoculation. Unexpectedly, many S. habrochaites plants were observed in which even multiplication of the Tm-1-resistance-breaking ToMV mutant LT1 was inhibited. An amino acid change in the positively selected region of the Tm-1 protein was responsible for the inhibition of LT1 multiplication. This amino acid change allowed Tm-1 to bind LT1 replication proteins without losing the ability to bind replication proteins of wild-type ToMV. The antiviral spectra and biochemical properties suggest that Tm-1 has evolved by changing the strengths of its inhibitory activity rather than diversifying the recognition spectra. In the LT1-resistant S. habrochaites plants inoculated with LT1, mutant viruses emerged whose multiplication was not inhibited by the Tm-1 allele that confers resistance to LT1. However, the resistance-breaking mutants were less competitive than the parental strains in the absence of Tm-1. Based on these results, we discuss possible coevolutionary processes of ToMV and Tm-1.