Johan A. Van Pelt

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.

NPR1 Modulates Cross-Talk between Salicylate- and Jasmonate-Dependent Defense Pathways through a Novel Function in the Cytosol

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signal transduction pathways in which salicylic acid (SA) and jasmonic acid (JA) play key roles. In this study, we investigated the molecular mechanism of the antagonistic effect of SA on JA signaling. Arabidopsis plants unable to accumulate SA produced 25-fold higher levels of JA and showed enhanced expression of the JA-responsive genes LOX2, PDF1.2, and VSP in response to infection by Pseudomonas syringae pv tomato DC3000, indicating that in wild-type plants, pathogen-induced SA accumulation is associated with the suppression of JA signaling. Analysis of the Arabidopsis mutant npr1, which is impaired in SA signal transduction, revealed that the antagonistic effect of SA on JA signaling requires the regulatory protein NPR1. Nuclear localization of NPR1, which is essential for SA-mediated defense gene expression, is not required for the suppression of JA signaling, indicating that cross-talk between SA and JA is modulated through a novel function of NPR1 in the cytosol.

Signal Signature and Transcriptome Changes of Arabidopsis During Pathogen and Insect Attack

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signaling pathways in which salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play key roles. To understand how plants integrate pathogen- and insect-induced signals into specific defense responses, we monitored the dynamics of SA, JA, and ET signaling in Arabidopsis after attack by a set of microbial pathogens and herbivorous insects with different modes of attack. Arabidopsis plants were exposed to a pathogenic leaf bacterium (Pseudomonas syringae pv. tomato), a pathogenic leaf fungus (Alternaria brassicicola), tissue-chewing caterpillars (Pieris rapae), cell-content-feeding thrips (Frankliniella occidentalis), or phloem-feeding aphids (Myzus persicae). Monitoring the signal signature in each plant-attacker combination showed that the kinetics of SA, JA, and ET production varies greatly in both quantity and timing. Analysis of global gene expression profiles demonstrated that the signal signature characteristic of each Arabidopsis-attacker combination is orchestrated into a surprisingly complex set of transcriptional alterations in which, in all cases, stress-related genes are overrepresented. Comparison of the transcript profiles revealed that consistent changes induced by pathogens and insects with very different modes of attack can show considerable overlap. Of all consistent changes induced by A. brassicicola, Pieris rapae, and E occidentalis, more than 50% also were induced consistently by P. syringae. Notably, although these four attackers all stimulated JA biosynthesis, the majority of the changes in JA-responsive gene expression were attacker specific. All together, our study shows that SA, JA, and ET play a primary role in the orchestration of the plants defense response, but other regulatory mechanisms, such as pathway cross-talk or additional attacker-induced signals, eventually shape the highly complex attacker-specific defense response.

Differential Effectiveness of Salicylate-Dependent and Jasmonate/Ethylene-Dependent Induced Resistance in Arabidopsis

Salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are each involved in the regulation of basal resistance against different pathogens. These three signals play important roles in induced resistance as well. SA is a key regulator of pathogen-induced systemic acquired resistance (SAR), whereas JA and ET are required for rhizobacteria-mediated induced systemic resistance (ISR). Both types of induced resistance are effective against a broad spectrum of pathogens. In this study, we compared the spectrum of effectiveness of SAR and ISR using an oomycete, a fungal, a bacterial, and a viral pathogen. In noninduced Arabidopsis plants, these pathogens are primarily resisted through either SA-dependent basal resistance (Peronospora parasitica and Turnip crinkle virus [TCV]), JA/ET-dependent basal resistance responses (Alternaria brassicicola), or a combination of SA-, JA-, and ET-dependent defenses (Xanthomonas campestris pv. armoraciae). Activation of ISR resulted in a significant level of protection against A. brassicicola, whereas SAR was ineffective against this pathogen. Conversely, activation of SAR resulted in a high level of protection against P. parasitica and TCV, whereas ISR conferred only weak and no protection against P. parasitica and TCV, respectively. Induction of SAR and ISR was equally effective against X. campestris pv. armoraciae. These results indicate that SAR is effective against pathogens that in noninduced plants are resisted through SA-dependent defenses, whereas ISR is effective against pathogens that in noninduced plants are resisted through JA/ET-dependent defenses. This suggests that SAR and ISR constitute a reinforcement of extant SA- or JA/ET-dependent basal defense responses, respectively.

Silencing of the Mitogen-Activated Protein Kinase MPK6 Compromises Disease Resistance in Arabidopsis

Here, we use a loss-of-function approach to demonstrate that the Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinase (MAPK) MPK6 plays a role in resistance to certain pathogens. MPK6-silenced Arabidopsis showed no apparent morphological phenotype or reduced fertility, indicating MPK6 is not required for development. However, resistances to an avirulent strain of Peronospora parasitica and avirulent and virulent strains of Pseudomonas syringae were compromised, suggesting that MPK6 plays a role in both resistance gene–mediated and basal resistance. Furthermore, this result demonstrates that MPK6s function cannot be fully complemented by other endogenous MAPKs. Although MPK6-silenced plants exhibited enhanced disease susceptibility, their ability to develop systemic acquired resistance or induced systemic resistance was unaffected. Expression of the pathogen-inducible gene VEGETATIVE STORAGE PROTEIN1 (VSP1) in MPK6-silenced plants was severalfold lower than in control plants, but the expression of other defense genes was comparable to the level observed in control plants. Taken together, these results provide direct evidence that a specific MAPK positively regulates VSP1 expression and resistance to a primary infection by certain pathogens, whereas systemic resistance and expression of several other defense genes appears to be mediated either by a functionally redundant MAPK(s) or independently from MPK6-dependent resistance.

Differential effectiveness of microbially induced resistance against herbivorous insects in Arabidopsis

Rhizobacteria-induced systemic resistance (ISR) and pathogen-induced systemic acquired resistance (SAR) have a broad, yet partly distinct, range of effectiveness against pathogenic microorganisms. Here, we investigated the effectiveness of ISR and SAR in Arabidopsis against the tissue-chewing insects Pieris rapae and Spodoptera exigua. Resistance against insects consists of direct defense, such as the production of toxins and feeding deterrents and indirect defense such as the production of plant volatiles that attract carnivorous enemies of the herbivores. Wind-tunnel experiments revealed that ISR and SAR did not affect herbivore-induced attraction of the parasitic wasp Cotesia rubecula (indirect defense). By contrast, ISR and SAR significantly reduced growth and development of the generalist herbivore S. exigua, although not that of the specialist P. rapae. This enhanced direct defense against S. exigua was associated with potentiated expression of the defense-related genes PDF1.2 and HEL. Expression profiling using a dedicated cDNA microarray revealed four additional, differentially primed genes in microbially induced S. exigua-challenged plants, three of which encode a lipid-transfer protein. Together, these results indicate that microbially induced plants are differentially primed for enhanced insect-responsive gene expression that is associated with increased direct defense against the generalist S. exigua but not against the specialist P. rapae.

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.

Colonization of Arabidopsis roots by Pseudomonas fluorescens primes the plant to produce higher levels of ethylene upon pathogen infection

Plants develop an enhanced defensive capacity against a broad spectrum of plant pathogens after colonization of the roots by selected strains of non-pathogenic, fluorescent Pseudomonas spp. In Arabidopsis thaliana, this rhizobacteria-induced systemic resistance (ISR) functions independently of salicylic acid but requires responsiveness to the plant hormones jasmonic acid and ethylene. Leaves of plants of which the roots are colonized by ISR-inducing Pseudomonas fluorescens WCS417r bacteria show an enhanced capacity to convert the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) to ethylene. Here we show that this enhanced ACC-converting capacity leads to a potentiated expression of the ethylene-responsive genes PDF1·2 and HEL after treatment of the leaves with 1 mM ACC, and a significantly higher level of ethylene emission after challenge inoculation with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000/avrRpt2. P. fluorescens WCS374r bacteria that are unable to induce ISR against P. syringae pv. tomato DC3000 in Arabidopsis likewise enhanced the in vivo ACC oxidase acitivity in Col-0 plants. Moreover, the ISR-compromised mutants jar1-1 and npr1-1 also showed a significant increase in their ability to convert ACC to ethylene after treatment of the roots with P. fluorescens WCS417r. These results suggest that the induction of an enhanced ACC-converting capacity is a general response of plants to P. fluorescens bacteria and that this response does not contribute to ISR against P. syringae pv. tomato DC3000 in Arabidopsis. Nevertheless, P. fluorescens strains clearly prime the plant to produce more ethylene upon pathogen infection. The increased capacity for ethylene production might contribute to an enhanced defensive capacity against pathogens that are sensitive to ethylene-dependent defense responses.

Transcriptome dynamics of Arabidopsis during sequential biotic and abiotic stresses

In nature, plants have to cope with a wide range of stress conditions that often occur simultaneously or in sequence. To investigate how plants cope with multi-stress conditions, we analyzed the dynamics of whole-transcriptome profiles of Arabidopsis thaliana exposed to six sequential double stresses inflicted by combinations of: (i) infection by the necrotrophic fungus Botrytis cinerea, (ii) herbivory by chewing larvae of Pieris rapae, and (iii) drought stress. Each of these stresses induced specific expression profiles over time, in which one-third of all differentially expressed genes was shared by at least two single stresses. Of these, 394 genes were differentially expressed during all three stress conditions, albeit often in opposite directions. When two stresses were applied in sequence, plants displayed transcriptome profiles that were very similar to the second stress, irrespective of the nature of the first stress. Nevertheless, significant first-stress signatures could be identified in the sequential stress profiles. Bioinformatic analysis of the dynamics of co-expressed gene clusters highlighted specific clusters and biological processes of which the timing of activation or repression was altered by a prior stress. The first-stress signatures in second stress transcriptional profiles were remarkably often related to responses to phytohormones, strengthening the notion that hormones are global modulators of interactions between different types of stress. Because prior stresses can affect the level of tolerance against a subsequent stress (e.g. prior herbivory strongly affected resistance to B. cinerea), the first-stress signatures can provide important leads for the identification of molecular players that are decisive in the interactions between stress response pathways.

Rhizobacterial volatiles and photosynthesis-related signals coordinate MYB72 expression in Arabidopsis roots during onset of induced systemic resistance and iron-deficiency responses

Summary In Arabidopsis roots, the transcription factor MYB72 plays a dual role in the onset of rhizobacteria‐induced systemic resistance (ISR) and plant survival under conditions of limited iron availability. Previously, it was shown that MYB72 coordinates the expression of a gene module that promotes synthesis and excretion of iron‐mobilizing phenolic compounds in the rhizosphere, a process that is involved in both iron acquisition and ISR signaling. Here, we show that volatile organic compounds (VOCs) from ISR‐inducing Pseudomonas bacteria are important elicitors of MYB72. In response to VOC treatment, MYB72 is co‐expressed with the iron uptake‐related genes FERRIC REDUCTION OXIDASE 2 (FRO2) and IRON‐REGULATED TRANSPORTER 1 (IRT1) in a manner that is dependent on FER‐LIKE IRON DEFICIENCY TRANSCRIPTION FACTOR (FIT), indicating that MYB72 is an intrinsic part of the plants iron‐acquisition response that is typically activated upon iron starvation. However, VOC ‐induced MYB72 expression is activated independently of iron availability in the root vicinity. Moreover, rhizobacterial VOC‐mediated induction of MYB72 requires photosynthesis‐related signals, while iron deficiency in the rhizosphere activates MYB72 in the absence of shoot‐derived signals. Together, these results show that the ISR‐ and iron acquisition‐related transcription factor MYB72 in Arabidopsis roots is activated by rhizobacterial volatiles and photosynthesis‐related signals, and enhances the iron‐acquisition capacity of roots independently of the iron availability in the rhizosphere. This work highlights the role of MYB72 in plant processes by which root microbiota simultaneously stimulate systemic immunity and activate the iron‐uptake machinery in their host plants.