Research programme on the genetic processes that allow the establishment of bacterial pathogens in their hosts

Project 1: Ax21 from Xanthomonas - its role in quorum sensing and as an elicitor of plant defense.
The Gram-negative bacterium Xanthomonas oryzae pv. oryzae causes bacterial leaf blight of rice, a devastating bacterial disease affecting rice plants in most rice-cultivating areas. Genetic resistances proved as being most efficient in controlling the disease, among them the resistance gene Xa21 (Song et al. 1995, Science 270: 1804-1806). Previous work had indicated that Ax21, a short peptide derived from a secreted protein of Xanthomonas, might trigger a defense response in rice upon perception by the receptor-like kinase Xa21, ultimately leading to plant resistance (Lee et al. 2009, Science 326: 850-853). Since this view was recently challenged, experiments were performed to elucidate the role of Ax21 as a trigger of plant defense. Wild-type bacteria and mutants in ax21 and raxST were inoculated into Xa21-transgenic rice plants (TP309-XA21) and symptoms were recorded. Surprisingly, the ax21 mutant of X. oryzae pv. oryzae could not overcome Xa21-mediated resistance, while the raxST mutant (rax = required for AvrXa21 activity) could overcome resistance, as reported by da Silva et al. in 2004 (Mol. Plant Microbe Interact. 17: 593-601). It is possible that Ax21 is only one of two or more ligands that are involved in Xa21-mediated resistance. In order to gain new insight into the pathogenicity of X. oryzae pv. oryzae, several strains were selected and sent for genome sequencing using Illumina technology.
Project 2: Banana Xanthomonas Wilt - an emergent pathogen in East Africa
Banana Xanthomonas Wilt (BXW), caused by pathogenic strains of Xanthomonas vasicola pv. musacearum (syn. X. campestris pv. musaceraum), is a new epidemic disease in several East African countries, incl. Uganda, Kenya, Congo, Rwanda and Burundi (Tripathi et al. 2009, Plant Disease 93: 440-451). Transgenic Xa21 banana plants have been reported to provide protection from Xanthomonas infection (Leena Tripathi, pers. commun.). We therefore decided to study the BXW pathogen. For comparative genomics, three strains isolated from enset plants and originating from Ethopia, the probable source of inoculum of the current outbreak in East and Central Africa, have been sent for sequencing using Illumina technology. We established a protocol to genetically modify X. vasicola pv. musacearum by DNA transformation (via electroporation). This technique will allow us to study the Xanthomonas-banana pathosystem in greater detail. The IRD laboratory had previously identified a candidate peptide, RapX, that could be secreted from xanthomonads via the type I secretion system (the Rax system) which is also involved in triggering Xa21-mediated resistance in rice (Guillaume Robin 2010, PhD thesis, University Montpellier 2, France). It was of interest to see whether or not this peptide is present in X. vasicola pv. musacearum. 12 strains of X. vasicola pv. musacearum originating from Ethopia, Uganda, Congo, Rwanda and Tanzania from the French collection of bacterial plant pathogens (http://www-intranet.angers.inra.fr/cfbp/) were chosen for study. Upon PCR amplification using primers annealing to raxST and raxA, DNA amplicons were sequenced. All 12 strains encoded the same double glycine leader peptide, MSRHVAWQRTAARRRHYRAGGQHGRASAAAGG, the mature part of which (QHGRASAAAGG) is identical to that from castor oil plant pathogens and closely related to that from xanthomonads infecting cassava, mango and beans, among others. We were also interested to see whether or not X. vasicola pv. musacearum encodes TAL (Transcription Activator Like) type III effectors, a major virulence factor of xanthomonads infecting rice and Citrus. Using a hybridization probe derived from X. oryzae pv. oryzae, Southern blot analyses did not shown any signals for TAL effector genes in the 12 strains of X. vasicola pv. musacearum. Also PCR using TAL effector-specific primers from X. oryzae pv. oryzae did not provide evidence for the existence of TAL effectors in X. vasicola pv. musacearum. While still not decisive since TAL effector genes from X. vasicola pv. musacearum could have sequence polymorphisms to escape detection via Southern blots and PCR, our findings are nevertheless corroborated by draft genome sequences which do not include candidate TAL effector genes in their assemblies.