María Luisa Pérez-Bueno

Imaging viral infection: studies on Nicotiana benthamiana plants infected with the pepper mild mottle tobamovirus

We have studied by kinetic Chl-fluorescence imaging (Chl-FI) Nicotiana benthamiana plants infected with the Italian strain of the pepper mild mottle tobamovirus (PMMoV-I). We have mapped leaf photosynthesis at different points of the fluorescence induction curve as well as at different post-infection times. Images of different fluorescence parameters were obtained to investigate which one could discriminate control from infected leaves in the absence of symptoms. The non-photochemical quenching (NPQ) of excess energy in photosystem II (PSII) seems to be the most adequate chlorophyll fluorescence parameter to assess the effect of tobamoviral infection on the chloroplast. Non-symptomatic mature leaves from inoculated plants displayed a very characteristic time-varying NPQ pattern. In addition, a correlation between NPQ amplification and virus localization by tissue-print was found, suggesting that an increase in the local NPQ values is associated with the areas invaded by the pathogen. Changes in chloroplast ultrastructure in non-symptomatic leaf areas showing different NPQ levels were also investigated. A gradient of ultrastructural modifications was observed among the different areas.

Spatial and temporal dynamics of primary and secondary metabolism in Phaseolus vulgaris challenged by Pseudomonas syringae

Many defense mechanisms contribute to the plant immune system against pathogens, involving the regulation of different processes of the primary and secondary metabolism. At the same time, pathogens have evolved mechanisms to hijack the plant defense in order to establish the infection and proliferate. Localization and timing of the host response are essential to understand defense mechanisms and resistance to pathogens (Rico et al. 2011). Imaging techniques, such as fluorescence imaging and thermography, are a very valuable tool providing spatial and temporal information about a series of plant processes. In this study, bean plants challenged with two pathovars of Pseudomonas syringae have been investigated. Pseudomonas syringae pv. phaseolicola 1448A and P. syringae pv. tomato DC3000 elicit a compatible and incompatible interaction in bean, respectively. Both types of host-pathogen interaction triggered different changes in the activity of photosynthesis and the secondary metabolism. We conclude that the combined analysis of leaf temperature, chlorophyll fluorescence and green fluorescence emitted by phenolics allows to discriminate compatible from incompatible P. syringae-Phaseolus vulgaris interactions in very early times of the infection, prior to the development of symptoms. These can constitute disease signatures that would allow an early identification of emerging plagues in crops.

Metabolic responses of avocado plants to stress induced by Rosellinia necatrix analysed by fluorescence and thermal imaging

One of the most important soilborne diseases affecting avocado (Persea americana Mill.) crops is white root rot, caused by the fungus Rosellinia necatrix. In this study we investigated the metabolic responses elicited by white root rot in the aerial part of the plant with special focus on the potential applications of imaging technique (including chlorophyll fluorescence (Chl-F), blue-green fluorescence and thermography) in early detection of the disease on leaves. The results show that leaf metabolism was significantly affected by the infection only when symptoms started to appear, which was probably related to the loss of root functionality. However, changes in some Chl-F parameters provided early indications of stress even prior to the development of symptoms. We suggest that the combinatorial analysis of several Chl-F parameters could be used as a method for early detection of stress related to white root rot, and might prove useful as a general indicator of biotic and abiotic stress in avocado plants.

Alterations in primary and secondary metabolism in Vitis vinifera ‘Malvasía de Banyalbufar’ upon infection with Grapevine leafroll‐associated virus 3

Plant defense mechanisms against pathogens result in differential regulation of various processes of primary and secondary metabolism. Imaging techniques, such as fluorescence imaging and thermography, are very valuable tools providing spatial and temporal information about these processes. In this study, effects of Grapevine leafroll-associated virus 3 (GLRaV-3) on grapevine physiology were analyzed in pot-grown asymptomatic plants of the white cultivar Malvasía de Banyalbufar. The virus triggered changes in the activity of photosynthesis and secondary metabolism. There was a decrease in the photorespiratory intermediates glycine and serine in infected plants, possibly as a defense response against the infection. The content of malate, which plays an important role in plant metabolism, also decreased. These results correlate with the increased non-photochemical quenching found in infected plants. On the other hand, the concentration of flavonols (represented by myricetin, kaempferol and quercetin derivatives) and hydroxycinnamic acids (which include derivatives of caffeic acid) increased following infection by the virus. These compounds could be responsible for the increase in multicolor fluorescence F440 (blue fluorescence) and F520 (green fluorescence) on the leaves, and changes in the fluorescence parameters F440/F680, F440/F740, F520/F680, F520/F740 and F680/F740. The combined analysis of chlorophyll fluorescence kinetics and blue-green fluorescence emitted by phenolics could constitute disease signatures allowing the discrimination between GLRaV-3 infected and non-infected plants at very early stage of infection, prior to the development of symptoms.

Contribution of the non-effector members of the HrpL regulon, iaaL and matE, to the virulence of Pseudomonas syringae pv. tomato DC3000 in tomato plants

BackgroundThe phytohormone indole-3-acetic acid (IAA) is widely distributed among plant-associated bacteria. Certain strains of the Pseudomonas syringae complex can further metabolize IAA into a less biologically active amino acid conjugate, 3-indole-acetyl-ε-L-lysine, through the action of the iaaL gene. In P. syringae and Pseudomonas savastanoi strains, the iaaL gene is found in synteny with an upstream gene, here called matE, encoding a putative MATE family transporter. In P. syringae pv. tomato (Pto) DC3000, a pathogen of tomato and Arabidopsis plants, the HrpL sigma factor controls the expression of a suite of virulence-associated genes via binding to hrp box promoters, including that of the iaaL gene. However, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear.ResultsA conserved hrp box motif is found upstream of the iaaL gene in the genomes of P. syringae strains. However, although the promoter region of matE is only conserved in genomospecies 3 of this bacterial group, we showed that this gene also belongs to the Pto DC3000 HrpL regulon. We also demonstrated that the iaaL gene is transcribed both independently and as part of an operon with matE in this pathogen. Deletion of either the iaaL or the matE gene resulted in reduced fitness and virulence of Pto DC3000 in tomato plants. In addition, we used multicolor fluorescence imaging to visualize the responses of tomato plants to wild-type Pto DC3000 and to its ΔmatE and ΔiaaL mutants. Activation of secondary metabolism prior to the development of visual symptoms was observed in tomato leaves after bacterial challenges with all strains. However, the observed changes were strongest in plants challenged by the wild-type strain, indicating lower activation of secondary metabolism in plants infected with the ΔmatE or ΔiaaL mutants.ConclusionsOur results provide new evidence for the roles of non-type III effector genes belonging to the Pto DC3000 HrpL regulon in virulence.

Picturing pathogen infection in plants

Abstract 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.

Temporal and Spatial Resolution of Activated Plant Defense Responses in Leaves of Nicotiana benthamiana Infected with Dickeya dadantii

The necrotrophic bacteria Dickeya dadantii is the causal agent of soft-rot disease in a broad range of hosts. The model plant Nicotiana benthamiana, commonly used as experimental host for a very broad range of plant pathogens, is susceptible to infection by D. dadantii. The inoculation with D. dadantii at high dose seems to overcome the plant defense capacity, inducing maceration and death of the tissue, although restricted to the infiltrated area. By contrast, the output of the defense response to low dose inoculation is inhibition of maceration and limitation in the growth, or even eradication, of bacteria. Responses of tissue invaded by bacteria (neighboring the infiltrated areas after 2–3 days post-inoculation) included: (i) inhibition of photosynthesis in terms of photosystem II efficiency; (ii) activation of energy dissipation as non-photochemical quenching in photosystem II, which is related to the activation of plant defense mechanisms; and (iii) accumulation of secondary metabolites in cell walls of the epidermis (lignins) and the apoplast of the mesophyll (phytoalexins). Infiltrated tissues showed an increase in the content of the main hormones regulating stress responses, including abscisic acid, jasmonic acid, and salicylic acid. We propose a mechanism involving the three hormones by which N. benthamiana could activate an efficient defense response against D. dadantii.

PsbO, PsbP, and PsbQ of photosystem II are encoded by gene families in Nicotiana benthamiana. Structure and functionality of their isoforms

The extrinsic proteins of photosystem II in plants (PsbO, PsbP and PsbQ) are known to be targets of stress. In previous work, differential regulation of hypothetical isoforms of these proteins was observed in Nicotiana benthamiana upon viral infection. Each of these proteins is encoded by a multigene family in this species: there are at least four genes encoding PsbO and PsbP and two encoding PsbQ. The results of structural and functional analyses suggest that PsbO and PsbP isoforms could show differences in activity, based on significant substitutions in their primary structure. Two psbQ sequences were isolated which encode identical mature proteins.

Multicolor Fluorescence Imaging as a Candidate for Disease Detection in Plant Phenotyping

The negative impact of conventional farming on environment and human health make improvements on farming management mandatory. Imaging techniques are implemented in remote sensing for monitoring crop fields and plant phenotyping programs. The increasingly large size and complexity of the data obtained by these techniques, makes the implementation of powerful mathematical tools necessary in order to identify informative parameters and to apply them in precision agriculture. Multicolor fluorescence imaging is a useful approach for the study of plant defense responses to stress factors at bench scale. However, it has not been fully applied to plant phenotyping. This work evaluates the possible application of multicolor fluorescence imaging in combination with thermography for the particular case of zucchini plants affected by soft-rot, caused by Dickeya dadantii. Several statistical models -based on logistic regression analysis (LRA) and artificial neural networks (ANN)- were obtained for the experimental system zucchini-D. dadantii, which classify new samples as “healthy” or “infected.” The LRA worked best in identifying high dose-infiltrated leaves (in infiltrated and non-infiltrated areas) whereas ANN offered a higher accuracy at identifying low dose-infiltrated areas. To assess the applicability of these results to cucurbits in a more general way, these models were validated for melon infected by the same pathogen, achieving accurate predictions for the infiltrated areas. The values of accuracy achieved are comparable to those found in the literature for classifiers identifying other infections based on data obtained by different techniques. Thus, MCFI in combination with thermography prove useful at providing data at lab scale that can be analyzed by machine learning. This approach could be scaled up to be applied in plant phenotyping.

Transcriptional profiles of Arabidopsis stomataless mutants reveal developmental and physiological features of life in the absence of stomata.

Loss of function of the positive stomata development regulators SPCH or MUTE in Arabidopsis thaliana renders stomataless plants; spch-3 and mute-3 mutants are extreme dwarfs, but produce cotyledons and tiny leaves, providing a system to interrogate plant life in the absence of stomata. To this end, we compared their cotyledon transcriptomes with that of wild-type plants. K-means clustering of differentially expressed genes generated four clusters: clusters 1 and 2 grouped genes commonly regulated in the mutants, while clusters 3 and 4 contained genes distinctively regulated in mute-3. Classification in functional categories and metabolic pathways of genes in clusters 1 and 2 suggested that both mutants had depressed secondary, nitrogen and sulfur metabolisms, while only a few photosynthesis-related genes were down-regulated. In situ quenching analysis of chlorophyll fluorescence revealed limited inhibition of photosynthesis. This and other fluorescence measurements matched the mutant transcriptomic features. Differential transcriptomes of both mutants were enriched in growth-related genes, including known stomata development regulators, which paralleled their epidermal phenotypes. Analysis of cluster 3 was not informative for developmental aspects of mute-3. Cluster 4 comprised genes differentially up−regulated in mute−3, 35% of which were direct targets for SPCH and may relate to the unique cell types of mute−3. A screen of T-DNA insertion lines in genes differentially expressed in the mutants identified a gene putatively involved in stomata development. A collection of lines for conditional overexpression of transcription factors differentially expressed in the mutants rendered distinct epidermal phenotypes, suggesting that these proteins may be novel stomatal development regulators. Thus, our transcriptome analysis represents a useful source of new genes for the study of stomata development and for characterizing physiology and growth in the absence of stomata.