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Transmission of xanthomonads to Medicago truncatula seeds

ABSTRACT: Plant pathogenic bacteria disseminate and survive through transmission to and by seeds of hosts and non-hosts plants. To investigate the interaction between xanthomonads and developing seeds of Medicago truncatula, plants at the ﬂower bud stage were spray inoculated until runoff with xanthomonads suspensions. Using the Medicago NimbleGen chip, a transcriptomic analysis was performed on seeds to characterize the molecular dialogue between Xanthomonas campestris pv. campestris in an incompatible situation with M. truncatula seeds and Xanthomonas alfalfae pv. alfalfae in a compatible situation at two developmental time points (16 and 32 days atfter pollination (DAP). Six-condition experiment, 16dap_Mock versus 16dap_Xaa, 16dap_Mock versus 16dap_Xcc, 32dap_Mock versus 32dap_Xaa, 32dap_Mock versus 32dap_Xcc. Biological replicates: 6 controls (16dap_Mock, 32dap_Mock), 12 treatments (16dap_Xaa, 16dap_Xcc, 32dap_Xaa, 32dap_Xcc), independently grown and harvested. One replicate per array.

ORGANISM(S): Medicago truncatula

SUBMITTER: Emmanuel Terrasson Marie-Agnès Jacques Sandra Pelletier Olivier Leprince Julia Buitink

PROVIDER: E-GEOD-49347 | ArrayExpress | 2013-07-31

SECONDARY ACCESSION(S): GSE49347PRJNA213709

REPOSITORIES: GEO, ArrayExpress

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Project description:A xanthomonad differential medium (designated Xan-D medium) was developed, on which streaks and colonies of xanthomonads, including 13 species of the genus Xanthomonas, turned wet-shining yellow-green and were surrounded with a smaller milky zone and a bigger clear zone in 3 to 4 days. The characteristics could easily be differentiated from those of yellow nonxanthomonads and other bacteria. The mechanism of color change and formation of a milky zone on the medium are mainly due to the Tween 80 hydrolytic capacity of xanthomonads. The gene, estA, responsible for Tween 80 hydrolysis was cloned and expressed in Escherichia coli, which acquired a capacity to hydrolyze Tween 80 and could turn green and form a milky zone on the Xan-D medium. The nucleotide sequence of estA is highly conserved in the xanthomonads, and the sequence was used to design a specific PCR primer set. The PCR amplification using the primer set amplified a 777-bp specific DNA fragment for all xanthomonad strains tested. The Xan-D medium was used to isolate and differentiate Xanthomonas campestris pv. campestris from naturally infected cabbages with black rot symptoms for a rapid diagnosis. All isolated X. campestris pv. campestris strains developed characteristic colonies and were positive in the PCR with the estA primer set. The Xan-D medium was further amended with antibiotics and successfully used for the detection of viable X. campestris pv. campestris cells from plant seeds. Although some yellow nonxanthomonads and other saprophytic bacteria from plant seeds could still grow on the medium, they did not interfere with the color development of X. campestris pv. campestris. However, Stenotrophomonas maltophilia, which is closely related to xanthomonads, existing in a seed lot could also develop yellow-green color but had different colony morphology and was negative in the PCR with the estA primer set. Accordingly, the combination of the Xan-D medium with the estA-specific PCR is a useful and reliable method for the isolation and detection of viable xanthomonad cells from plant materials.

Project description:ABSTRACT The pathogenic bacterium Xanthomonas campestris pv. campestris, the causal agent of black rot of Brassicaceae, manipulates the physiology and the innate immunity of its hosts. Association genetic and reverse-genetic analyses of a world panel of 45 X. campestris pv. campestris strains were used to gain understanding of the genetic basis of the bacterium's pathogenicity to Arabidopsis thaliana. We found that the compositions of the minimal predicted type III secretome varied extensively, with 18 to 28 proteins per strain. There were clear differences in aggressiveness of those X. campestris pv. campestris strains on two Arabidopsis natural accessions. We identified 3 effector genes (xopAC, xopJ5, and xopAL2) and 67 amplified fragment length polymorphism (AFLP) markers that were associated with variations in disease symptoms. The nature and distribution of the AFLP markers remain to be determined, but we observed a low linkage disequilibrium level between predicted effectors and other significant markers, suggesting that additional genetic factors make a meaningful contribution to pathogenicity. Mutagenesis of type III effectors in X. campestris pv. campestris confirmed that xopAC functions as both a virulence and an avirulence gene in Arabidopsis and that xopAM functions as a second avirulence gene on plants of the Col-0 ecotype. However, we did not detect the effect of any other effector in the X. campestris pv. campestris 8004 strain, likely due to other genetic background effects. These results highlight the complex genetic basis of pathogenicity at the pathovar level and encourage us to challenge the agronomical relevance of some virulence determinants identified solely in model strains. IMPORTANCE The identification and understanding of the genetic determinants of bacterial virulence are essential to be able to design efficient protection strategies for infected plants. The recent availability of genomic resources for a limited number of pathogen isolates and host genotypes has strongly biased our research toward genotype-specific approaches. Indeed, these do not consider the natural variation in both pathogens and hosts, so their applied relevance should be challenged. In our study, we exploited the genetic diversity of Xanthomonas campestris pv. campestris, the causal agent of black rot on Brassicaceae (e.g., cabbage), to mine for pathogenicity determinants. This work evidenced the contribution of known and unknown loci to pathogenicity relevant at the pathovar level and identified these virulence determinants as prime targets for breeding resistance to X. campestris pv. campestris in Brassicaceae.

			Action	DRS
	E-GEOD-49347.idf.txt	Idf
	E-GEOD-49347.processed.1.zip	Processed
	E-GEOD-49347.raw.1.zip	Raw
	E-GEOD-49347.sdrf.txt	Txt

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Transmission of xanthomonads to Medicago truncatula seeds

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