Leendert C. van Loon

A Novel Signaling Pathway Controlling Induced Systemic Resistance in Arabidopsis

Plants have the ability to acquire an enhanced level of resistance to pathogen attack after being exposed to specific biotic stimuli. In Arabidopsis, nonpathogenic, root-colonizing Pseudomonas fluorescens bacteria trigger an induced systemic resistance (ISR) response against infection by the bacterial leaf pathogen P. syringae pv tomato. In contrast to classic, pathogen-induced systemic acquired resistance (SAR), this rhizobacteria-mediated ISR response is independent of salicylic acid accumulation and pathogenesis-related gene activation. Using the jasmonate response mutant jar1, the ethylene response mutant etr1, and the SAR regulatory mutant npr1, we demonstrate that signal transduction leading to P. fluorescens WCS417r–mediated ISR requires responsiveness to jasmonate and ethylene and is dependent on NPR1. Similar to P. fluorescens WCS417r, methyl jasmonate and the ethylene precursor 1-aminocyclopropane-1-carboxylate were effective in inducing resistance against P. s. tomato in salicylic acid–nonaccumulating NahG plants. Moreover, methyl jasmonate–induced protection was blocked in jar1, etr1, and npr1 plants, whereas 1-aminocyclopropane-1-carboxylate–induced protection was affected in etr1 and npr1 plants but not in jar1 plants. Hence, we postulate that rhizobacteria-mediated ISR follows a novel signaling pathway in which components from the jasmonate and ethylene response are engaged successively to trigger a defense reaction that, like SAR, is regulated by NPR1. We provide evidence that the processes downstream of NPR1 in the ISR pathway are divergent from those in the SAR pathway, indicating that NPR1 differentially regulates defense responses, depending on the signals that are elicited during induction of resistance.

Impact of root exudates and plant defense signaling on bacterial communities in the rhizosphere. A review

Despite significant advances in crop protection, plant diseases cause a 20% yield loss in food and cash crops worldwide. Therefore, interactions between plants and pathogens have been studied in great detail. In contrast, the interplay between plants and non-pathogenic microorganisms has received scant attention, and differential responses of plants to pathogenic and non-pathogenic microorganisms are as yet not well understood. Plants affect their rhizosphere microbial communities that can contain beneficial, neutral and pathogenic elements. Interactions between the different elements of these communities have been studied in relation to biological control of plant pathogens. One of the mechanisms of disease control is induced systemic resistance (ISR). Studies on biological control of plant diseases have focused on ISR the last decade, because ISR is effective against a wide range of pathogens and thus offers serious potential for practical applications in crop protection. Such applications may however affect microbial communities associated with plant roots and interfere with the functioning of the root microbiota. Here, we review the possible impact of plant defense signaling on bacterial communities in the rhizosphere. To better assess implications of shifts in the rhizosphere microflora we first review effects of root exudates on soil microbial communities. Current knowledge on inducible defense signaling in plants is discussed in the context of recognition and systemic responses to pathogenic and beneficial microorganisms. Finally, the as yet limited knowledge on effects of plant defense on rhizosphere microbial communities is reviewed and we discuss future directions of research that will contribute to unravel the molecular interplay of plants and their beneficial microflora.

Determinants of Pseudomonas putida WCS358 involved in inducing systemic resistance in plants

SUMMARY Pseudomonas putida WCS358 is a plant growth-promoting rhizobacterium originally isolated from the rhizosphere of potato. It can suppress soil-borne plant diseases by siderophore-mediated competition for iron, but it has also been reported to result in induced systemic resistance (ISR) in Arabidopsis thaliana. Bacterial determinants of this strain involved in inducing systemic resistance in Arabidopsis were investigated using a Tn5 transposon mutant defective in biosynthesis of the fluorescent siderophore pseudobactin, a non-motile Tn5 mutant lacking flagella, and a spontaneous phage-resistant mutant lacking the O-antigenic side chain of the lipopolysaccharides (LPS). When using Pseudomonas syringae pv. tomato as the challenging pathogen, purified pseudobactin, flagella and LPS all triggered ISR. However, the mutants were all as effective as the parental strain, suggesting redundancy in ISR-triggering traits in WCS358. The Botrytis cinerea-tomato, B. cinerea-bean and Colletotrichum lindemuthianum-bean model systems were used to test further the potential of P. putida WCS358 to induce ISR. Strain WCS358 significantly reduced disease development in all three systems, indicating that also on tomato and bean WCS358 can trigger ISR. In both tomato and bean, the LPS mutant had lost the ability to induce resistance, whereas the flagella mutant was still effective. In bean, the pseudobactin mutant was still effective, whereas this mutant has lost its effectivity in tomato. In both bean and tomato, flagella isolated from the parental strain were not effective, whereas LPS or pseudobactin did induce systemic resistance.

Effect of Genetically Modified Pseudomonas putida WCS358r on the Fungal Rhizosphere Microflora of Field-Grown Wheat

ABSTRACT We released genetically modified Pseudomonas putidaWCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the phz biosynthetic gene locus of strainP. fluorescens 2-79 and constitutively produced the antifungal compound phenazine-1-carboxylic acid (PCA). In the springs of 1997 and 1998 we sowed wheat seeds treated with either GMM 2, GMM 8, or WCS358r (approximately 107 CFU per seed), and measured the numbers, composition, and activities of the rhizosphere microbial populations. During both growing seasons, all three bacterial strains decreased from 107 CFU per g of rhizosphere sample to below the limit of detection (102 CFU per g) 1 month after harvest of the wheat plants. The phz genes were stably maintained, and PCA was detected in rhizosphere extracts of GMM-treated plants. In 1997, but not in 1998, fungal numbers in the rhizosphere, quantified on 2% malt extract agar (total filamentous fungi) and on Komadas medium (mainly Fusarium spp.), were transiently suppressed in GMM 8-treated plants. We also analyzed the effects of the GMMs on the rhizosphere fungi by using amplified ribosomal DNA restriction analysis. Introduction of any of the three bacterial strains transiently changed the composition of the rhizosphere fungal microflora. However, in both 1997 and 1998, GMM-induced effects were distinct from those of WCS358r and lasted for 40 days in 1997 and for 89 days after sowing in 1998, whereas effects induced by WCS358r were detectable for 12 (1997) or 40 (1998) days. None of the strains affected the metabolic activity of the soil microbial population (substrate-induced respiration), soil nitrification potential, cellulose decomposition, plant height, or plant yield. The results indicate that application of GMMs engineered to have improved antifungal activity can exert nontarget effects on the natural fungal microflora.

Induced systemic resistance in Arabidopsis thaliana against Pseudomonas syringae pv. tomato by 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens.

Pseudomonas fluorescens strains that produce the polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are among the most effective rhizobacteria that suppress root and crown rots, wilts, and damping-off diseases of a variety of crops, and they play a key role in the natural suppressiveness of some soils to certain soilborne pathogens. Root colonization by 2,4-DAPG-producing P. fluorescens strains Pf-5 (genotype A), Q2-87 (genotype B), Q8r1-96 (genotype D), and HT5-1 (genotype N) produced induced systemic resistance (ISR) in Arabidopsis thaliana accession Col-0 against bacterial speck caused by P. syringae pv. tomato. The ISR-eliciting activity of the four bacterial genotypes was similar, and all genotypes were equivalent in activity to the well-characterized strain P. fluorescens WCS417r. The 2,4-DAPG biosynthetic locus consists of the genes phlHGF and phlACBDE. phlD or phlBC mutants of Q2-87 (2,4-DAPG minus) were significantly reduced in ISR activity, and genetic complementation of the mutants restored ISR activity back to wild-type levels. A phlF regulatory mutant (overproducer of 2,4-DAPG) had ISR activity equivalent to the wild-type Q2-87. Introduction of DAPG into soil at concentrations of 10 to 250 μM 4 days before challenge inoculation induced resistance equivalent to or better than the bacteria. Strain Q2-87 induced resistance on transgenic NahG plants but not on npr1-1, jar1, and etr1 Arabidopsis mutants. These results indicate that the antibiotic 2,4-DAPG is a major determinant of ISR in 2,4-DAPG-producing P. fluorescens, that the genotype of the strain does not affect its ISR activity, and that the activity induced by these bacteria operates through the ethylene- and jasmonic acid-dependent signal transduction pathway.

Early Responses of Tobacco Suspension Cells to Rhizobacterial Elicitors of Induced Systemic Resistance

Colonization of roots by selected strains of fluorescent Pseudomonas spp. can trigger induced systemic resistance (ISR) against foliar pathogens in a plant species-specific manner. It has been suggested that early responses in cell suspension cultures in response to rhizobacterial elicitors, such as generation of active oxygen species (AOS) and extracellular medium alkalinization (MA), are linked to the development of ISR in whole plants. Perception of flagellin was demonstrated to elicit ISR in Arabidopsis, and bacterial lipopolysaccharides (LPS) have been shown to elicit several defense responses and to act as bacterial determinants of ISR in various plant species. In the present study, the LPS-containing cell walls, the pyoverdine siderophores, and the flagella of Pseudomonas putida WCS358, P. fluorescens WCS374, and P. fluorescens WCS417, which are all known to act as elicitors of ISR in selected plant species, were tested for their effects on the production of AOS, MA, elevation of cytoplasmic Ca(2+) ([Ca(2+)](cyt)), and defense-related gene expression in tobacco suspension cells. The LPS of all three strains, the siderophore of WCS374, and the flagella of WCS358 induced a single, transient, early burst of AOS, whereas the siderophores of WCS358 and WCS417 and the flagella of WCS374 and WCS417 did not. None of the compounds caused cell death. Once stimulated by the active compounds, the cells became refractory to further stimulation by any of the active elicitors, but not to the elicitor cryptogein from the oomycete Phytophthora cryptogea, indicating that signaling upon perception of the different rhizobacterial compounds rapidly converges into a common response pathway. Of all compounds tested, only the siderophores of WCS358 and WCS417 did not induce MA; the flagella of WCS374 and WCS417, although not active as elicitors of AOS, did induce MA. These results were corroborated by using preparations from relevant bacterial mutants. The active rhizobacterial elicitors led to a rapid increase in [Ca(2+)](cyt), peaking at 6 min, whereas the inactive siderophores of WCS358 and WCS417 elicited a single spike at 1 min. Elicitation of the cells by cell-wall LPS of WCS358 or the siderophore of WCS374 induced a weak, transient expression of several defense-related genes, including PAL and GST. The spectrum of early responses of the suspension cells was not matched by the expression of ISR in whole tobacco plants against Erwinia carotovora pv. carotovora. Of the live bacterial strains, only WCS358 elicited significant ISR, but application of the LPS or the siderophore of all three strains also elicited ISR. Notably, the absence of elicitation of AOS and MA in suspension-cultured cells but induction of ISR in whole plants by the siderophore of WCS358, which was lost upon treatment with the siderophore-minus mutant of WCS358, indicates that the early responses in suspension cells are not predictive of the ability to induce ISR in whole plants. Possible explanations for these discrepancies are discussed.

Effects of Pseudomonas putida modified to produce phenazine-1-carboxylic acid and 2,4-diacetylphloroglucinol on the microflora of field grown wheat

Pseudomonas putida WCS358r, genetically modified to have improved activity against soil-borne pathogens, was released into the rhizosphere of wheat. Two genetically modified derivatives carried the phzor the phl biosynthetic gene loci and constitutively produced either the antifungal compound phenazine-1-carboxylic acid (PCA) or the antifungal and antibacterial compound 2,4-diacetylphloroglucinol (DAPG). In 1997 and 1998, effects of single introductions of PCA producing derivatives on the indigenous microflora were studied. A transient shift in the composition of the total fungal microflora, determined by amplified ribosomal DNA restiction analysis (ARDRA), was detected. Starting in 1999, effects of repeated introduction of genetically modified microorganisms (GMMs) were studied. Wheat seeds coated with the PCA producer, the DAPG producer, a mixture of the PCA and DAPG producers, or WCS358r, were sown and the densities, composition and activities of the rhizosphere microbial populations were measured. All introduced strains decreased from 107CFU per gram of rhizosphere sample to below the detection limit after harvest of the wheat plants. The phz genes were stably maintained in the PCA producers, and PCA was detected in rhizosphere extracts of plants treated with this strain or with the mixture of the PCA and DAPG producers. The phl genes were also stably maintained in the DAPG producing derivative of WCS358r. Effects of the genetically modified bacteria on the rhizosphere fungi and bacteria were analyzed by using amplified ribosomal DNA restriction analysis. Introduction of the genetically modified bacterial strains caused a transient change in the composition of the rhizosphere microflora. However, introduction of the GMMs did not affect the several soil microbial activities that were investigated in this study.

Iron‐regulated metabolites produced by Pseudomonas fluorescens WCS374r are not required for eliciting induced systemic resistance against Pseudomonas syringae pv. tomato in Arabidopsis

The plant growth‐promoting rhizobacterium Pseudomonas fluorescens WCS374r produces several iron‐regulated metabolites, including the fluorescent siderophore pseudobactin (Psb374), salicylic acid (SA), and pseudomonine (Psm), a siderophore that contains a SA moiety. After purification of Psb374 from culture supernatant of WCS374r, its structure was determined following isoelectrofocusing and tandem mass spectrometry, and found to be identical to the fluorescent siderophore produced by P. fluorescens ATCC 13525. To study the role of SA and Psm production in colonization of Arabidopsis thaliana roots and in induced systemic resistance (ISR) against Pseudomonas syringae pv. tomato (Pst) by strain WCS374r, mutants disrupted in the production of these metabolites were obtained by homologous recombination. These mutants were further subjected to transposon Tn5 mutagenesis to generate mutants also deficient in Psb374 production. The mutants behaved similar to the wild type in both their Arabidopsis rhizosphere‐colonizing capacity and their ability to elicit ISR against Pst. We conclude that Psb374, SA, and Psm production by P. fluorescens WCS374r are not required for eliciting ISR in Arabidopsis.

The Intelligent Behavior of Plants

Plants are as adept as animals and humans in reacting effectively to their ever-changing environment. Of necessity, their sessile nature requires specific adaptations, but their cells possess a network-type communication system with emerging properties at the level of the organ or entire plant. The specific adjustments in growth and development of plants can be taken to represent behavior. Their ability to learn from experience and to memorize previous experiences in order to optimize fitness allows effective acclimation to environmental stresses and can be considered a form of intelligence. Intelligent behavior is exemplified by the exceptional versatility of plants to deal with abiotic stresses as well as microbial and insect attack by balancing appropriate defensive reactions.