Transcriptomics and knockout mutant analysis of rhizobacteria-mediated induced systemic resistance in Arabidopsis

A classic example of induced resistance is triggered after infection by a necrotizing pathogen, rendering uninfected,distal parts more resistant to subsequent pathogen attack, and is often referred to as systemic acquired resistance (SAR). A phenotypically comparable type of induced resistance is triggered after root colonization of plants by selected strains of non-pathogenic Pseudomonas spp., and is often called induced systemic resistance (ISR). In contrast to pathogen-induced SAR, rhizobacteria-mediated ISR is not associated with accumulation of SA. Instead, rhizobacteria-mediated ISR requires the hormonal signals jasmonic acid (JA) and ethylene (ET). Whereas pathogen-induced SAR is accompanied by an increase in PR gene expression throughout the plant, rhizobacteria-mediated ISR in Arabidopsis is not associated with a systemic increase in the expression of any known defense-related genes. To identify ISR-related genes that are specifically induced after treatment of the roots with ISR-triggering Pseudomonas fluorescens WCS417r bacteria, a large collection of gene trap and enhancer trap lines of Arabidopsis was screened on the basis of inducible expression of the ß-glucuronidase (GUS) gene. This resulted in the identification of the AtTLP1 gene, encoding a thaumatin-like protein that belongs to the PR-5 family of PR proteins. Further analysis revealed that this gene is specifically expressed in xylem tissue of WCS417r-colonized roots. The expression of the AtTLP1 gene was also induced after treatment of the roots with resistance inducing Pseudomonas putida WCS358r and P. fluorescens WCS374r, but not after treatment with Escherichia coli. Moreover, the AtTLP1 gene was also responsive to the ET precursor 1-aminocyclopropane-1-carboxylate (ACC) but not to SA or JA, indicating that the expression of this gene is regulated by ET. AtTLP1 over-expressing and knockout mutant plants showed normal levels of WCS417r-mediated ISR against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Hence, it must be concluded that WCS417r-induced expression of AtTLP1 in the roots is a common response of Arabidopsis to colonization of the roots by Pseudomonas bacteria and is not related to the induction of ISR. In another attempt to identify ISR-related genes, the transcriptional profile of over 8,000 Arabidopsis genes during the local onset of ISR in the roots and the systemic state and expression of ISR in the leaves was surveyed using Affymetrix Arabidopsis GeneChips. Root colonization by WCS417r elicited a significant change in the local expression of 97 genes. Among these were genes predicted to be involved in cell rescue and defense (18%), metabolism (14%), regulation of gene transcription (15%), and cellular communication and signal transduction (7%). Systemically in the leaves, prior to challenge inoculation, none of the 8,000 genes tested showed a consistent change in expression in response to colonization of the roots by the ISR-inducing WCS417r bacteria. This indicates that the state of ISR in the leaves is not associated with detectable changes i n gene expression, even though these leaves are clearly more resistant to subsequent pathogen attack. However, after challenge inoculation with Pst DC3000, 81 genes showed an augmented expression pattern in leaves expressing ISR, suggesting that these genes were primed to respond faster and/or more strongly to pathogen attack. Among these primed genes were genes involved in transcription, e.g. EREBP2 and a WRKY family transcription factor gene, as well as genes involved in defense, such as PDF1.2 and a gene encoding a thaumatin-like protein. The majority of the primed genes were shown to be regulated in a JA- and/or ET-dependent manner. Priming of pathogen-induced genes allows the plant to react more effectively to an invading pathogen, which might explain the br oad-spectrum action of rhizobacteria-mediated ISR. Moreover, since mainly JA/ET-dependent genes were primed, this can explain why ISR is predominantly effective against pathogens that are resisted by JA/ET-dependent defense responses in Arabidopsis. To investigate the role of some of the genes that were specifically induced in the roots upon colonization by WCS417r, knockout mutant nalysis of a subset of these genes was performed . This revealed that most of the genes with altered expression patterns in the roots were not of major importance for the onset of ISR. However, a knockout mutant with a T-DNA insertion 15 basepairs downstream of the start codon of the R2R3-MYB-like transcription factor gene AtMYB72, appeared to be blocked in its ability to express ISR against Pst DC3000. This block in ISR induction in this myb72 mutant was apparent after treatment with live WCS417r bacteria, crude cell walls of WCS417r, as well as live WCS358r bacteria. Analysis of the expression of AtMYB72 revealed that it is not only induced by ISR-inducing WCS417r, but also by ACC. WCS417r-induced expression of AtMYB72 was blocked in the ET-insensitive, ISR-defective mutant ein2-1, but not in the ISR-responsive, SA-defective transformant NahG. Moreover, the AtMYB72 protein was found to physically interact in vitro with the ET-regulatory protein EIL3. Together, these results indicate that AtMYB72 plays an essential role in the local onset of ISR in the roots and that its expression is regulated by the ET signaling pathway