Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene, AvrLmS-Lep2

SummaryLeptosphaeria maculans, the causal agent of blackleg disease, interacts with Brassica napus (oilseed rape, canola) in a gene-for-gene manner. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in the cultivar Surpass 400. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (Bulked-Segregant-Sequencing) to rapidly identify one candidate avirulence gene present in the effector repertoire of L. maculans. AvrLep2 was cloned using a bi-parental cross of avirulent and virulent L. maculans isolates and a classical map-based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400 and Topas-LepR2. The gene renamed AvrLmS-Lep2 encodes for a small cysteine-rich protein of unknown function with an N-terminal secretory signal peptide, which are common features of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS-Lep2 / LepR2 interaction phenotype was found to vary from a typical hypersensitive response to intermediate resistance sometimes at the edge of, or evolving toward, susceptibility depending on the inoculation conditions. AvrLmS-Lep2 was nevertheless sufficient to significantly reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field.

Gtgen3A, a novel plant GH3 β-glucosidase, modulates gentio-oligosaccharide metabolism in Gentiana

Gentiobiose, a β-1,6-linked glycosyl-disaccharide, accumulates abundantly in Gentianaceae and is involved in aspects of plant development, such as fruits ripening and release of bud dormancy. However, the mechanisms regulating the amount of gentio-oligosaccharide accumulation in plants remain obscure. The present study aimed to identify an enzyme that modulates gentio-oligosaccharide amount in gentian (Gentiana triflora). A protein responsible for gentiobiose hydrolysis, GtGen3A, was identified by partial purification and its peptide sequence analysis. The enzyme had a molecular mass of ∼67 kDa without a secretory signal peptide sequence. Sequence analysis revealed that GtGen3A could be a β-glucosidase member belonging to glycoside hydrolase family 3 (GH3). GtGen3A showed a homology to GH3 β-glucan exohydrolases, ExoI of Hordeum vulgare, and ExgI from Zea mays, which preferentially hydrolyzed β-1,3- and β-1,4-linked oligosaccharides. The purified recombinant GtGen3A (rGtGen3A) produced in Escherichia coli showed optimal reaction at pH 6.5 and 20°C. The rGtGen3A liberated glucose from β-1,2-, β-1,3-, β-1,4-, and β-1,6-linked oligosaccharides, and showed the highest activity toward gentiotriose among the substrates tested. Kinetic analysis also revealed that rGtGen3A preferentially hydrolyzed gentiotriose. Virus-induced gene silencing of Gtgen3A in gentian plantlets resulted in predominant accumulation of gentiotriose rather than gentiobiose. Furthermore, the expression level of Gtgen3A was almost similar to the amount of gentiobiose in field-grown gentians. These findings suggest that the main function of GtGen3A is the hydrolysis of gentiotriose to gentiobiose, and that GtGen3A plays a role in modulating gentiobiose amounts in gentian.

Construction of Non-Antibiotic Resistance Expression Vector by Secretory β-Gal as a Selective Marker in Lactobacillus

On account of the spreading problem of resistance gene and off-target effects of functional gene products expressed by bacteria, pMG36e, a plasmid with the erythromycin- resistance (Emr) gene, which could shuttle between Lactobacillus and E. coli, was reconstructed into a non-antibiotic secretory expression vector with the beta-Galactosidase (β-Gal) gene for selection, then linked a secretory signal peptide sequence to the non-antibiotic resistance vector, thus the secretory non-antibiotic resistance vector was constructed. Finally, to test the vector's function, the SO7 gene of Eimeria tenella (E. tenella) was cloned into the vector and expressed successfully. This system may be used as a model to explore the feasibility of vaccine vectors using the β-Gal gene for selection, and collect fundamental data for expressing other homogeneous and heterogeneous functional genes in such a novel vector.