Increased resistance against citrus canker mediated by a citrus mitogen-activated protein kinase.

Mitogen-activated protein kinases (MAPK) play crucial roles in plant immunity. We previously identified a citrus MAPK (CsMAPK1) as a differentially expressed protein in response to infection by Xanthomonas aurantifolii, a bacterium that causes citrus canker in Mexican lime but a hypersensitive reaction in sweet oranges. Here, we confirm that, in sweet orange, CsMAPK1 is rapidly and preferentially induced by X. aurantifolii relative to Xanthomonas citri. To investigate the role of CsMAPK1 in citrus canker resistance, we expressed CsMAPK1 in citrus plants under the control of the PR5 gene promoter, which is induced by Xanthomonas infection and wounding. Increased expression of CsMAPK1 correlated with a reduction in canker symptoms and a decrease in bacterial growth. Canker lesions in plants with higher CsMAPK1 levels were smaller and showed fewer signs of epidermal rupture. Transgenic plants also revealed higher transcript levels of defense-related genes and a significant accumulation of hydrogen peroxide in response to wounding or X. citri infection. Accordingly, nontransgenic sweet orange leaves accumulate both CsMAPK1 and hydrogen peroxide in response to X. aurantifolii but not X. citri infection. These data, thus, indicate that CsMAPK1 functions in the citrus canker defense response by inducing defense gene expression and reactive oxygen species accumulation during infection.

The Xanthomonas citri effector protein PthA interacts with citrus proteins involved in nuclear transport, protein folding and ubiquitination associated with DNA repair.

Xanthomonas axonopodis pv. citri utilizes the type III effector protein PthA to modulate host transcription to promote citrus canker. PthA proteins belong to the AvrBs3/PthA family and carry a domain comprising tandem repeats of 34 amino acids that mediates protein-protein and protein-DNA interactions. We show here that variants of PthAs from a single bacterial strain localize to the nucleus of plant cells and form homo- and heterodimers through the association of their repeat regions. We hypothesize that the PthA variants might also interact with distinct host targets. Here, in addition to the interaction with alpha-importin, known to mediate the nuclear import of AvrBs3, we describe new interactions of PthAs with citrus proteins involved in protein folding and K63-linked ubiquitination. PthAs 2 and 3 preferentially interact with a citrus cyclophilin (Cyp) and with TDX, a tetratricopeptide domain-containing thioredoxin. In addition, PthAs 2 and 3, but not 1 and 4, interact with the ubiquitin-conjugating enzyme complex formed by Ubc13 and ubiquitin-conjugating enzyme variant (Uev), required for K63-linked ubiquitination and DNA repair. We show that Cyp, TDX and Uev interact with each other, and that Cyp and Uev localize to the nucleus of plant cells. Furthermore, the citrus Ubc13 and Uev proteins complement the DNA repair phenotype of the yeast Deltaubc13 and Deltamms2/uev1a mutants, strongly indicating that they are also involved in K63-linked ubiquitination and DNA repair. Notably, PthA 2 affects the growth of yeast cells in the presence of a DNA damage agent, suggesting that it inhibits K63-linked ubiquitination required for DNA repair.

Transcriptional analysis of the sweet orange interaction with the citrus canker pathogens Xanthomonas axonopodis pv. citri and Xanthomonas axonopodis pv. aurantifolii.

Xanthomonas axonopodis pv. citri (Xac) and Xanthomonas axonopodis pv. aurantifolii pathotype C (Xaa) are responsible for citrus canker disease; however, while Xac causes canker on all citrus varieties, Xaa is restricted to Mexican lime, and in sweet oranges it triggers a defence response. To gain insights into the differential pathogenicity exhibited by Xac and Xaa and to survey the early molecular events leading to canker development, a detailed transcriptional analysis of sweet orange plants infected with the pathogens was performed. Using differential display, suppressed subtractive hybridization and microarrays, we identified changes in transcript levels in approximately 2.0% of the approximately 32,000 citrus genes examined. Genes with altered expression in response to Xac/Xaa surveyed at 6 and 48 h post-infection (hpi) were associated with cell-wall modifications, cell division and expansion, vesicle trafficking, disease resistance, carbon and nitrogen metabolism, and responses to hormones auxin, gibberellin and ethylene. Most of the genes that were commonly modulated by Xac and Xaa were associated with basal defences triggered by pathogen-associated molecular patterns, including those involved in reactive oxygen species production and lignification. Significantly, we detected clear changes in the transcriptional profiles of defence, cell-wall, vesicle trafficking and cell growth-related genes in Xac-infected leaves between 6 and 48 hpi. This is consistent with the notion that Xac suppresses host defences early during infection and simultaneously changes the physiological status of the host cells, reprogramming them for division and growth. Notably, brefeldin A, an inhibitor of vesicle trafficking, retarded canker development. In contrast, Xaa triggered a mitogen-activated protein kinase signalling pathway involving WRKY and ethylene-responsive transcriptional factors known to activate downstream defence genes.