Bernard Dumas

Cytological, genetic, and molecular analysis to characterize compatible and incompatible interactions between Medicago truncatula and Colletotrichum trifolii.

In this study, a new pathosystem was established using the model plant Medicago truncatula and Colletotrichum trifolii, the causal agent of anthracnose on Medicago sativa. Screening of a few M. truncatula lines identified Jemalong and F83005.5 as resistant and susceptible to Colletotrichum trifolii race 1, respectively. Symptom analysis and cytological studies indicated that resistance of Jemalong was associated with a hypersensitive response of the plant. The two selected lines were crossed, and inoculations with C. trifolii were performed on the resulting F1 and F2 progenies. Examination of the disease phenotypes indicated that resistance was dominant and was probably due to a major resistance gene. Molecular components of the resistance were analyzed through macroarray experiments. Expression profiling of 126 expressed sequence tags corresponding to 92 genes, which were selected for their putative functions in plant defense or signal transduction, were compared in Jemalong and F83005.5 lines. A strong correlation was observed between the number of up-regulated genes and the resistance phenotype. Large differences appeared at 48 h postinoculation; more than 40% of the tested genes were up-regulated in the Jemalong line compared with only 10% in the susceptible line. Interestingly, some nodulin genes were also induced in the resistant line upon inoculation with C. trifolii.

Regulation of enzymes involved in lignin biosynthesis: induction of O-methyltransferase mRNAs during the hypersensitive reaction of tobacco to tobacco mosaic virus.

The mRNAs encoding orthodiphenol-O-methyltransferases (OMTs; EC 2.1.1.6), which are involved in the biosynthesis of lignin precursors, are highly induced in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus (TMV). OMT messengers were fractionated on a sucrose gradient and translated in vitro. Protein A-Sepharose columns adsorbed with specific antisera raised against purified OMTs were used to select translation products, and the translatable activity of OMT mRNA was measured at different stages of infection. Oligonucleotides derived from peptide sequences of purified OMT I were used to prime polymerase chain reactions; total RNA was used as template to allow the isolation of an OMT I clone. RNA blots, hybridized with the OMT I probe, revealed a unique messenger of 1.7 kb. The kinetics of accumulation of OMT I mRNAs during the hypersensitive reaction to TMV parallels the kinetics of translation and suggests that an increase in mRNA controls the increase in the rate of enzyme synthesis. In healthy plants, RNA blot hybridization showed that the steady-state level of OMT I mRNA is very high in vascular tissue compared to the level measured in leaves.

Ulvan, a Sulfated Polysaccharide from Green Algae, Activates Plant Immunity through the Jasmonic Acid Signaling Pathway

The industrial use of elicitors as alternative tools for disease control needs the identification of abundant sources of them. We report on an elicitor obtained from the green algae Ulva spp. A fraction containing most exclusively the sulfated polysaccharide known as ulvan-induced expression of a GUS gene placed under the control of a lipoxygenase gene promoter. Gene expression profiling was performed upon ulvan treatments on Medicago truncatula and compared to phytohormone effects. Ulvan induced a gene expression signature similar to that observed upon methyl jasmonate treatment (MeJA). Involvement of jasmonic acid (JA) in ulvan response was confirmed by detecting induction of protease inhibitory activity and by hormonal profiling of JA, salicylic acid (SA) and abscisic acid (ABA). Ulvan activity on the hormonal pathway was further consolidated by using Arabidopsis hormonal mutants. Altogether, our results demonstrate that green algae are a potential reservoir of ulvan elicitor which acts through the JA pathway.

Partial resistance of Medicago truncatula to Aphanomyces euteiches is associated with protection of the root stele and is controlled by a major QTL rich in proteasome-related genes.

A pathosystem between Aphanomyces euteiches, the causal agent of pea root rot disease, and the model legume Medicago truncatula was developed to gain insights into mechanisms involved in resistance to this oomycete. The F83005.5 French accession and the A17-Jemalong reference line, susceptible and partially resistant, respectively, to A. euteiches, were selected for further cytological and genetic analyses. Microscopy analyses of thin root sections revealed that a major difference between the two inoculated lines occurred in the root stele, which remained pathogen free in A17. Striking features were observed in A17 roots only, including i) frequent pericycle cell divisions, ii) lignin deposition around the pericycle, and iii) accumulation of soluble phenolic compounds. Genetic analysis of resistance was performed on an F7 population of 139 recombinant inbred lines and identified a major quantitative trait locus (QTL) near the top of chromosome 3. A second study, with near-isogenic line responses to A. euteiches confirmed the role of this QTL in expression of resistance. Fine-mapping allowed the identification of a 135-kb sequenced genomic DNA region rich in proteasome-related genes. Most of these genes were shown to be induced only in inoculated A17. Novel mechanisms possibly involved in the observed partial resistance are proposed.

Elicitor Activity of a Fungal Endopolygalacturonase in Tobacco Requires a Functional Catalytic Site and Cell Wall Localization

CLPG1, an endopolygalacturonase (endoPG) gene of Colletotrichum lindemuthianum, was transferred to tobacco (Nicotiana tabacum) leaves by using the Agrobacterium tumefaciens transient delivery system. The following four constructs were prepared:CLPG1, with or without its signal peptide (SP; PG1, PG1ΔSP); CLPG1 with the tobaccoexpansin1 SP instead of its own SP (Exp::PG1ΔSP); and a mutated version of the latter on two amino acids potentially involved in the catalytic site of CLPG1 (D202N/D203N). Chlorotic and necrotic lesions appeared 5 to 7 d postinfiltration, exclusively in response to CLPG1 fused to the expansin SP. The lesions were correlated to the production of an active enzyme. Necrosis-inducing activity, as well as endoPG activity, were completely abolished by site-directed mutagenesis. Ultrastructural immunocytolocalization experiments indicated that the expansin SP addressed CLPG1 to the cell wall. Staining of parenchyma cells revealed the progressive degradation of pectic material in junction zones and middle lamella as a function of time after infiltration, ultimately leading to cell separation. A 30% decrease in the GalUA content of the cell walls was simultaneously recorded, thereby confirming the hydrolytic effect of CLPG1 on pectic polysaccharides, in planta. The elicitor activity of CLPG1 was further illustrated by the induction of defense responses comprising active oxygen species and β-1,3-glucanase activity, before leaf necrosis. Altogether, the data demonstrate that an appropriate SP and a functional catalytic site are required for the proper expression and elicitor activity of the fungal endoPG CLPG1 in tobacco.

Functional analysis of CLPT1, a Rab/GTPase required for protein secretion and pathogenesis in the plant fungal pathogen Colletotrichum lindemuthianum.

In eukaryotic cells, Rab/GTPases are major regulators of vesicular trafficking and are involved in essential processes including exocytosis, endocytosis and cellular differentiation. To investigate the role of these proteins in fungal pathogenicity, a dominant-negative mutant allele of CLPT1, a Rab/GTPase of the bean pathogen Colletotrichum lindemuthianum, was expressed in transgenic strains. This mutated gene encodes the amino-acid substitution N123I analogous to the N133I substitution in a known trans-dominant inhibitor of the Sec4 Rab/GTPase from Saccharomyces cerevisiae. A pectinase gene promoter was used to drive the CLPT1(N123I) allele in C. lindemuthianum, allowing the expression of the foreign gene on pectin medium and during pathogenesis, but not on glucose. The same strategy was used to overexpress the wild-type CLPT1 allele. During growth on pectin medium, production of extracellular pectinases was strongly impaired only in CLPT1(N123I)-expressing strains. Cytological analysis revealed that CLPT1(N123I) strains accumulated intracellular aggregates only on pectin, resulting from the fusion of vesicles containing polysaccharides or glycoproteins. Moreover, these strains showed a severe reduction of pathogenesis and were unable to penetrate the host cells. These results indicated that the Rab/GTPase CLPT1 is essential for fungal pathogenesis by regulating the intracellular transport of secretory vesicles involved in the delivery of proteins to the extracellular medium and differentiation of infectious structures.

One-step purification and characterization of a lignin-specific O-methyltransferase from poplar

O-Methyltransferases (OMT; EC 2.1.1.6) play an important role in the synthesis of lignin precursors by catalyzing the O-methylation of o-diphenolic substrates such as caffeic acid (CA) and 5-hydroxyferulic acid (5OH). Here, we report on the purification of a lignin-specific OMT (38 kDa) from poplar (Populus trichocarpa x P. deltoides). The OMT was purified from xylem by a single affinity chromatography step on adenosine agarose. The enzyme uses both CA and 5OH as substrates. We previously have reported the cloning of a corresponding OMT cDNA [Dumas et al., Plant Physiol. 98 (1992) 796-797]. Expression of this OMT cDNA in Escherichia coli further confirmed the identity of the clone. Genomic hybridization demonstrates the presence of one or two OMT genes per haploid poplar genome. RNA gel blot hybridization shows high levels of steady-state OMT mRNA in the xylem of young poplar trees, as compared to the levels in leaves.

Purification of tobacco O-methyltransferases by affinity chromatography and estimation of the rate of synthesis of the enzymes during hypersensitive reaction to virus infection.

The three tobacco (Nicotiana tabacum L.) S-adenosyl-L-methionine: o-diphenol-O-methyltransferases (OMTs; EC 2.1.1.6) were purified to homogeneity by affinity chromatography on adenosine-agarose. Amounts and catalytic actities of the enzymes were measured in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus. The drastic increase in activity of each enzyme upon infection was shown to arise from the accumulation of enzymatic protein with constant specific enzymatic activity. Rates of OMT synthesis were determined from pulse-labeling experiments with L-[14C]leucine injected into the leaves. The specific radioactivities of the homogenous enzymes were compared in healthy and tobacco mosaic virus-infected tobacco. The results demonstrated that increase in OMT amounts is a consequence of de novo synthesis of the enzymes.

Host and Nonhost Resistance in Medicago–Colletotrichum Interactions

Medicago truncatula lines resistant (A17) or susceptible (F83005.5) to the alfalfa pathogen Colletotrichum trifolii were used to compare defense reactions induced upon inoculation with C. trifolii or with the nonadapted pathogens C. lindemuthianum and C. higginsianum. Nonadapted Colletotrichum spp. induced a hypersensitive response (HR)-like reaction similar to the one induced during the host-incompatible interaction. Molecular analyses indicated an induction of PR10 and chalcone synthase genes in host and nonhost interactions but delayed responses were observed in the F83005.5 line. The clste12 penetration-deficient C. lindemuthianum mutant induced an HR and defense gene expression, showing that perception of nonadapted strains occurs before penetration of epidermal cells. Cytological and transcriptomic analyses performed upon inoculation of near-isogenic M. truncatula lines, differing only at the C. trifolii resistance locus, Ct1, with the nonadapted Colletotrichum strain, showed that nonhost responses are similar in the two lines. These included a localized oxidative burst, accumulation of fluorescent compounds, and transient expression of a small number of genes. Host interactions were characterized by a group of defense and signaling-related genes induced at 3 days postinoculation, associated with an accumulation of salicylic acid. Together, these results show that M. truncatula displays a rapid and transient response to nonadapted Colletotrichum strains and that this response is not linked to the C. trifolii resistance locus.

Detection of nucleic acid–protein interactions in plant leaves using fluorescence lifetime imaging microscopy

DNA-binding proteins (DNA-BPs) and RNA-binding proteins (RNA-BPs) have critical roles in living cells in all kingdoms of life. Various experimental approaches exist for the study of nucleic acid–protein interactions in vitro and in vivo, but the detection of such interactions at the subcellular level remains challenging. Here we describe how to detect nucleic acid–protein interactions in plant leaves by using a fluorescence resonance energy transfer (FRET) approach coupled to fluorescence lifetime imaging microscopy (FLIM). Proteins of interest (POI) are tagged with a GFP and transiently expressed in plant cells to serve as donor fluorophore. After sample fixation and cell wall permeabilization, leaves are treated with Sytox Orange, a nucleic acid dye that can function as a FRET acceptor. Upon close association of the GFP-tagged POI with Sytox-Orange-stained nucleic acids, a substantial decrease of the GFP lifetime due to FRET between the donor and the acceptor can be monitored. Treatment with RNase before FRET–FLIM measurements allows determination of whether the POI associates with DNA and/or RNA. A step-by-step protocol is provided for sample preparation, data acquisition and analysis. We describe how to calibrate the equipment and include a tutorial explaining the use of the FLIM software. To illustrate our approach, we provide experimental procedures to detect the interaction between plant DNA and two proteins (the AeCRN13 effector from the oomycete Aphanomyces euteiches and the AtWRKY22 defensive transcription factor from Arabidopsis). This protocol allows the detection of protein–nucleic acid interactions in plant cells and can be completed in <2 d.