Successful intergeneric transfer of a major apple scab resistance gene (Rvi6) from apple to pear and precise comparison of the downstream molecular mechanisms of this resistance in both species.

BACKGROUND: Scab is the most important fungal disease of apple and pear. Apple (Malus x domestica Borkh.) and European pear (Pyrus communis L.) are genetically related but they are hosts of two different fungal species: Venturia inaequalis for apple and V. pyrina for European pear. The apple/V. inaequalis pathosystem is quite well known, whereas knowledge about the pear/V. pyrina pathosystem is still limited. The aim of our study was to analyse the mode of action of a major resistance gene of apple (Rvi6) in transgenic apple and pear plants interacting with the two scab species (V. inaequalis and V. pyrina), in order to determine the degree of functional transferability between the two pathosystems. RESULTS: Transgenic pear clones constitutively expressing the Rvi6 gene from apple were compared to a scab transgenic apple clone carrying the same construct. After inoculation in greenhouse with V. pyrina, strong defense reactions and very limited sporulation were observed on all transgenic pear clones tested. Microscopic observations revealed frequent aborted conidiophores in the Rvi6 transgenic pear / V. pyrina interaction. The macro- and microscopic observations were very comparable to the Rvi6 apple / V. inaequalis interaction. However, this resistance in pear proved variable according to the strain of V. pyrina, and one of the strains tested overcame the resistance of most of the transgenic pear clones. Comparative transcriptomic analyses of apple and pear resistant interactions with V. inaequalis and V. pyrina, respectively, revealed different cascades of molecular mechanisms downstream of the pathogen recognition by Rvi6 in the two species. Signal transduction was triggered in both species with calcium (and G-proteins in pear) and interconnected hormonal signaling (jasmonic acid in pear, auxins in apple and brassinosteroids in both species), without involvement of salicylic acid. This led to the induction of defense responses such as a remodeling of primary and secondary cell wall, lipids biosynthesis (galactolipids in apple and cutin and cuticular waxes in pear), systemic acquired resistance signal generation (in apple) or perception in distal tissues (in pear), and the biosynthesis of phenylpropanoids (flavonoids in apple but also lignin in pear). CONCLUSION: This study is the first example of a successful intergeneric transfer of a resistance gene among Rosaceae, with a resistance gene functioning towards another species of pathogen.

Membrane-targeted HrpNEa can modulate apple defense gene expression.

Fire blight caused by Erwinia amylovora is the major bacterial disease of tribe Maleae, including apple. Among the proteins secreted by this bacterium, HrpNEa, also called harpin, is known to induce hypersensitive response in nonhost plants and to form amyloid oligomers leading to pore opening in the plasma membrane and alteration of membrane homeostasis. To better understand the physiological effects of HrpNEa in the host plant, we produced transgenic apple plants expressing HrpNEa with or without a secretion signal peptide (SP). HrpNEa expressed with a SP was found to be associated within the membrane fraction, in accordance with amyloidogenic properties and the presence of transmembrane domains revealed by in silico analysis. Expression analysis of 28 apple defense-related genes revealed gene modulations in the transgenic line expressing membrane-targeted HrpNEa. While apple transgenic trees displaying a high constitutive expression level of SP-HrpNEa showed a slight reduction of infection frequency after E. amylovora inoculation, there was no decrease in the disease severity. Thus HrpNEa seems to act as an elicitor of host defenses, when localized in the host membrane.

Chitinases of Trichoderma atroviride Induce Scab Resistance and Some Metabolic Changes in Two Cultivars of Apple.

ABSTRACT This study reports the combination of a transgene-mediated defense mechanism with a conventionally bred resistance in order to improve apple scab resistance. Two cultivars of apple (Galaxy, scab-susceptible, and Ariane, carrying the Vf resistance gene) were transformed with endochitinase and exochitinase genes derived from the biocontrol fungus Trichoderma atroviride. The obtained transgenic lines were analyzed for the expression of both genes and resistance to two races of the pathogen Venturia inaequalis: the common race 1 and race 6 which overcomes the resistance conferred by the Vf gene. A negative correlation between growth of transgenic lines and endochitinase activity was observed. Reduced growth appeared to be associated with high lignin content and high peroxidase as well as glucanase activity, suggesting that endochitinase activity may disturb the metabolism of the plant. Scab inoculation with races 1 and 6 performed in a growth chamber on 14 lines of normal vigor identified 6 lines with significantly enhanced resistance. Ten lines with reduced vigor were tested in vitro with a bioassay on rooted shoots. All lines expressing high endochitinase activity exhibited a significant reduction of scab symptoms.

Strategies to improve plant resistance to bacterial diseases through genetic engineering.

Many different genetic strategies have been proposed to engineer plant resistance to bacterial diseases, including producing antibacterial proteins of non-plant origin, inhibiting bacterial pathogenicity or virulence factors, enhancing natural plant defenses and artificially inducing programmed cell death at the site of infection. These are based on our knowledge of the mechanisms of action of antibacterial compounds and of the successive steps in plant-bacterial interactions. This article presents the different approaches and demonstrates that, even though several of these ideas have already been applied, no commercial applications have yet been achieved.

EfficientAgrobacterium-mediated transformation and recovery of transgenic plants from pear (Pyrus communis L.).

An efficient and reproducible method was established for genetic transformation of one pear variety (Conferénce) usingAgrobacterium tumefaciens-mediated gene transfer. Wounded leaves of in vitro micropropagated plants were cocultivated with the disarmed strain EHA101 harbouring the binary vector pFAJ3000 carrying the chimaericnptII andgus genes. The protocol included a 3-6 month dark period on a regeneration medium solidified with gelrite, which contained 100 mg/l kanamycin. Up to 42% of inoculated leaves produced transformed buds or bud clusters. Expression, presence and integration of transgenes was confirmed by a histochemical test, polymerase chain reaction and Southern blot hybridisation, respectively. The transgenec plants could be successfully acclimatized in the glasshouse. This transformation procedure was also successfully applied to two other pear varieties, namely Doyenné du Cornice and Passe-Crassane, albeit at much lower transformation rates.

Organogenesis from 'Passe Crassane' and 'Old Home' pear (Pyrus communis L.) protoplasts and isoenzymatic trueness-to-type of the regenerated plants.

Large numbers of highly viable mesophyll protoplasts were isolated from shoot cultures of the scion cv 'Passe Crassane' and the rootstock genotype 'Old Home' of common pear (Pyrus communis L.). Protoplasts were cultured for both genotypes either as liquid layers or as liquid-over-agar cultures, in ammonium-free MS medium with 0.5 M mannitol, 50 mg/l casein enzymatic hydrolysate (CEH), 2.0 mg/l NAA and 1.0 mg/l BAP, plus either 0.5 mg/l IAA (for 'Old Home') or 2.0 mg/l IAA (for 'Passe Crassane'). Protoplast microcalli, obtained by day 60 ('Passe Crassane') or day 80 ('Old Home'), were transferred for further growth to ammonium-free MS medium with 2.0 mg/l NAA and 1.0 mg/l BAP. Shoot bud regeneration from the protoplastderived callus was first attempted between 100 ('Passe Crassane') and 120 ('Old Home') days after protoplast isolation. For 'Passe Crassane', shoot buds were regenerated (day 130) on a half-strength MS medium with 0.1 mg/l IBA, 0.5 mg/l BAP, 50 mg/l CEH and 20 mg/l Ca-panthotenate. For 'Old Home', shoot but regeneration only occurred 30 days later and on the same medium as above, which was additionally supplemented with double the concentration of the group B vitamins found in the original MS formulation and 0.05 mg/l GA3. Following micropropagation and in vitro rooting of shoots, the plants were transferred to soil following standard procedures. Trueness-to-type of the regenerated plants was assessed by analysing their leaf isozyme banding profiles (for EST, AP, PRX, SOD, ENP, LAP, PGI, AAT, ADH, MDH and PGM) and comparing them to those corresponding to the original shoots that provided the protoplasts. No differences between the mother shoots and the protoclones were observed for any one of the 11 isozyme systems studied.

Adventitious shoot regeneration from leaf tissue of three pear (Pyrus sp.) cultivars in vitro.

To develop an adventitious regeneration system for pear cultivars, several experiments were conducted with 2 cultivars of Pyrus communis L. ('Seckel' and 'Louise Bonne') and one cultivar of P. bretschneideri Rehd. ('Crystal Pear'). Half-leaves, taken from shoots proliferating on Lepoivre medium, were plated in petri-dishes on medium supplemented with various combinations of cytokinins and auxins. Cultures of the above cultivars had been established from mature trees. Among the growth regulators tested, thidiazuron (TDZ), combined with naphthalene-acetic acid (NAA), was the most efficient for stimulation of adventitious shoots. The optimum level of TDZ was about 3 uM; shoot regeneration was observed over a wide range of TDZ and NAA concentrations (0.5 to 5 uM and 2.5 to 13 um, respectively). Among different macronutrient compositions, 1/2 and 1/4 Murashige and Skoog were the most effective. Sucrose concentrations (10 to 50 g L-1) had a linear significant effect on shoot regeneration of 'Crystal Pear'.

Inheritance of pollen enzymes and polyploid origin of apple (Malus x domestica Borkh.).

Electrophoresis of 7 pollen enzymes was applied to 5 progenies from controlled crosses and one self-progeny of apple. Segregation data were examined according to three kinds of hypotheses: monogenic disomic, bigenic disomic and tetrasomic inheritance Twenty codominant alleles and a recessive null were identified. Results provided evidence of bigenic disomic inheritance in most cases: 6 pairs of homoeologous loci carrying identical homoeoalleles were revealed; only 2 enzymes exhibited a simple monogenic control. Preferential pairing between pairs of homologous chromosomes in meiosis can be postulated. These results indicated an allopolyploid origin of apple genome. Fixed heterozygosity occurred for several enzymes, which is a typical feature of allopolyploidy. Loss of duplicate gene expression can account for the monogenic control of 2 of the enzymes.