Caroline Labbé

Silicon Enhances the Accumulation of Diterpenoid Phytoalexins in Rice: A Potential Mechanism for Blast Resistance

ABSTRACT Although several reports underscore the importance of silicon (Si) in controlling Magnaporthe grisea on rice, no study has associated this beneficial effect with specific mechanisms of host defense responses against this fungal attack. In this study, however, we provide evidence that higher levels of momilactone phytoalexins were found in leaf extracts from plants inoculated with M. grisea and amended with silicon (Si(+)) than in leaf extracts from inoculated plants not amended with silicon (Si(-) ) or noninoculated Si(+) and Si(-) plants. On this basis, the more efficient stimulation of the terpenoid pathway in Si(+) plants and, consequently, the increase in the levels of momilactones appears to be a factor contributing to enhanced rice resistance to blast. This may explain the lower level of blast severity observed on leaves of Si(+) plants at 96 h after inoculation with M. grisea. The results of this study strongly suggest that Si plays an active role in the resistance of rice to blast rather than the formation of a physical barrier to penetration by M. grisea.

Antifungal Activity of Flocculosin, a Novel Glycolipid Isolated from Pseudozyma flocculosa

ABSTRACT Flocculosin, a glycolipid isolated from the yeast-like fungus Pseudozyma flocculosa, was investigated for in vitro antifungal activity. The compound displayed antifungal properties against several pathogenic yeasts. Synergistic activity was observed between flocculosin and amphotericin B, and no significant cytotoxicity was demonstrated when tested against human cell lines.

Effects of milsana and benzothiadiazole on the ultrastructure of powdery mildew haustoria on cucumber.

ABSTRACT Disease assessments and cytological investigations provided valuable information on the modes of action and efficacies of two prophylactic compounds, Milsana and benzothiadiazole (BTH), against powdery mildew development on long English cucumber. Milsana application significantly reduced disease incidence relative to inoculated controls through induction of localized resistance. Microscopic observations showed most haustoria had collapsed in the localized Milsana treatment and were encapsulated by an amorphous material impregnated by electron-opaque substances. The rapidity of haustorial collapse (within 4 days of treatment application) together with the encasement by electron-dense substances stained blue by toluidine blue O suggest that phenolics are possibly involved in the Milsana defense response. Cytochemical labeling of chitin with a wheat germ agglutinin/ovomucoid-gold complex showed that complete cellular disorganization of the fungus had occurred without disturbance to chitin in the walls of mycelia and haustoria. This may indicate that chitinolytic activity is not important in the Milsana-activated defense response. Application of high doses of BTH induced occasional cell wall thickening and accumulation of a compound that stained purple by toluidine blue O, but the defense response was weak, sporadic, and insufficient to reduce powdery mildew infection on cucumber. Responses to BTH could not be differentiated in terms of timing of the initial application or systemicity.

Identification and functional characterization of silicon transporters in soybean using comparative genomics of major intrinsic proteins in Arabidopsis and rice

Silicon (Si) confers several benefits to many plant species when absorbed as silicic acid through nodulin 26-like intrinsic proteins (NIPs). The NIPs belong to major intrinsic protein (MIP) family, members of which form channels with high selectivity to control transport of water and different solutes. Here, comparative genomic analysis of the MIPs was performed to investigate the presence of Si transporter MIPs in soybean. Thorough analysis of phylogeny, gene organization, transcriptome profiling and protein modeling was performed to characterize MIPs in rice, Arabidopsis and soybean. Based on several attributes, two putative Si transporter genes, GmNIP2-1 and GmNIP2-2, were identified, characterized and cloned from soybean. Expression of both genes was detected in shoot and root tissues, and decreased as Si increased. The protein encoded by GmNIP2-2 showed functionality for Si transport when expressed in Xenopus oocytes, thus confirming the genetic capability of soybean to absorb the element. Comparative analysis of MIPs in plants provides opportunities to decipher gene evolution, functionality and selectivity of nutrient uptake mechanisms. Exploitation of this strategy has helped to uncover unique features of MIPs in soybean. The identification and functional characterization of Si transporters can be exploited to optimize the benefits that plants can derive from Si absorption.

Insertional mutagenesis of a fungal biocontrol agent led to discovery of a rare cellobiose lipid with antifungal activity.

ABSTRACT Insertional mutagenesis was applied for the first time to a fungal biocontrol agent, Pseudozyma flocculosa, in an attempt to obtain mutants with altered antagonistic properties. Transformants were obtained via DNA-mediated transformation. Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci. The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties. Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods. Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2′,4′-diacetoxy-5′-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of P. flocculosa.

Discovery of a multigene family of aquaporin silicon transporters in the primitive plant Equisetum arvense

Plants benefit greatly from silicon (Si) absorption provided that they contain Si transporters. The latter have recently been identified in the roots of some higher plants known to accumulate high concentrations of Si, and all share a high level of sequence identity. In this study, we searched for transporters in the primitive vascular plant Equisetum arvense (horsetail), which is a valuable but neglected model plant for the study of Si absorption, as it has one of the highest Si concentrations in the plant kingdom. Our initial attempts to identify Si transporters based on sequence homology with transporters from higher plants proved unsuccessful, suggesting a divergent structure or property in horsetail transporters. Subsequently, through sequencing of the horsetail root transcriptome and a search using amino acid sequences conserved in plant aquaporins, we were able to identify a multigene family of aquaporin Si transporters. Comparison of known functional domains and phylogenetic analysis of sequences revealed that the horsetail proteins belong to a different group than higher-plant Si transporters. In particular, the newly identified proteins contain a STAR pore as opposed to the GSGR pore common to all previously identified Si transporters. In order to determine its functionality, the proteins were heterologously expressed in both Xenopus oocytes and Arabidopsis, and the results showed that the horsetail proteins are extremely efficient a transporting Si. These findings offer new insights into the elusive properties of Si and its absorption by plants.

A precise spacing between the NPA domains of aquaporins is essential for silicon permeability in plants.

The controversy surrounding silicon (Si) benefits and essentiality in plants is exacerbated by the differential ability of species to absorb this element. This property is seemingly enhanced in species carrying specific nodulin 26-like intrinsic proteins (NIPs), a subclass of aquaporins. In this work, our aim was to characterize plant aquaporins to define the features that confer Si permeability. Through comparative analysis of 985 aquaporins in 25 species with differing abilities to absorb Si, we were able to predict 30 Si transporters and discovered that Si absorption is exclusively confined to species that possess NIP-III aquaporins with a GSGR selectivity filter and a precise distance of 108 amino acids (AA) between the asparagine-proline-alanine (NPA) domains. The latter feature is of particular significance since it had never been reported to be essential for Si selectivity. Functionality assessed in the Xenopus oocyte expression system showed that NIPs with 108 AA spacing exhibited Si permeability, while proteins differing in that distance did not. In subsequent functional studies, a Si transporter from poplar mutated into variants with 109- or 107-AA spacing failed to import, and a tomato NIP gene mutated from 109 to 108 AA exhibited a rare gain of function. These results provide a precise molecular basis to classify higher plants into Si accumulators or excluders.

Phenolic compounds that confer resistance to spruce budworm

Phenolic compounds are apparently important in the defence mechanisms of conifers. To test the hypothesis that phenolic compounds in resistant white spruce [Picea glauca (Moench) Voss (Pinaceae)] impart resistance against spruce budworm [Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae)], we performed aqueous extractions of current‐year shoots of white spruce that were tolerant of varying levels of budworm defoliation. High‐performance liquid chromatographic profiles of water extracts of P. glauca needles differed between resistant and susceptible trees. Further nuclear magnetic resonance analyses identified two phenolic glucosides in susceptible white spruce, picein [3‐(β‐d‐glucosyloxy)‐hydroxy‐acetophenone] and pungenin [3‐(β‐d‐glucosyloxy)‐4‐hydroxy‐acetophenone], and two phenolics in resistant white spruce, pungenol (3′,4′‐hydroxy‐acetophenone) and piceol (4′‐hydroxyacetophenone). We focused on the performance of spruce budworm when piceol and pungenol were added to the diet. These two compounds significantly reduced larval survival, retarded development, and reduced pupal mass. Food consumption by sixth‐instar spruce budworms was affected by a combination of the phenolic compounds. These results suggest that the two phenolic compounds reduce the pressure of spruce budworm herbivory on specific host tree phenotypes. Thus, the mechanism of defence in P. glauca apparently reflects a strategy of constitutive resistance.

Suppression of induced resistance in cucumber through disruption of the flavonoid pathway

ABSTRACT In this study, cucumber plants (Cucumis sativus) expressing induced resistance against powdery mildew (caused by Podosphaera xanthii) were infiltrated with inhibitors of cinnamate 4-hydroxylase, 4-coumarate:CoA ligase (4CL), and chalcone synthase (CHS) to evaluate the role of flavonoid phytoalexin production in induced disease resistance. Light and transmission electron microscopy demonstrated ultrastructural changes in inhibited plants, and biochemical analyses determined levels of CHS and beta-glucosidase enzyme activity and 4CL protein accumulation. Our results showed that elicited plants displayed a high level of induced resistance. In contrast, down regulation of CHS, a key enzyme of the flavonoid pathway, resulted in nearly complete suppression of induced resistance, and microscopy confirmed the development of healthy fungal haustoria within these plants. Inhibition of 4CL ligase, an enzyme largely responsible for channeling phenylpropanoid metabolites into the lignin pathway, had little effect on induced disease resistance. Biochemical analyses revealed similar levels of 4CL protein accumulation for all treatments, suggesting no alterations of nontargeted functions within inhibited plants. Collectively, the results of this study support the idea that induced resistance in cucumber is largely correlated with rapid de novo biosynthesis of flavonoid phytoalexin compounds.

Silicon‐mediated resistance of Arabidopsis against powdery mildew involves mechanisms other than the salicylic acid (SA)‐dependent defence pathway

On absorption by plants, silicon (Si) offers protection against many fungal pathogens, including powdery mildews. The mechanisms by which Si exerts its prophylactic role remain enigmatic, although a prevailing hypothesis suggests that Si positively influences priming. Attempts to decipher Si properties have been limited to plants able to absorb Si, which excludes the model plant Arabidopsis because it lacks Si influx transporters. In this work, we were able to engineer Arabidopsis plants with an Si transporter from wheat (TaLsi1) and to exploit mutants (pad4 and sid2) deficient in salicylic acid (SA)-dependent defence responses to study their phenotypic response and changes in defence expression against Golovinomyces cichoracearum (Gc) following Si treatment. Our results showed that TaLsi1 plants contained significantly more Si and were significantly more resistant to Gc infection than control plants when treated with Si, the first such demonstration in a plant transformed with a heterologous Si transporter. The resistant plants accumulated higher levels of SA and expressed higher levels of transcripts encoding defence genes, thus suggesting a role for Si in the process. However, TaLsi1 pad4 and TaLsi1 sid2 plants were also more resistant to Gc than were pad4 and sid2 plants following Si treatment. Analysis of the resistant phenotypes revealed a significantly reduced production of SA and expression of defence genes comparable with susceptible controls. These results indicate that Si contributes to Arabidopsis defence priming following pathogen infection, but highlight that Si will confer protection even when priming is altered. We conclude that Si-mediated protection involves mechanisms other than SA-dependent defence responses.