Identification of a major QTL together with several minor additive or epistatic QTLs for resistance to fire blight in apple in two related progenies.

Although fire blight, caused by the bacterium Erwinia amylovora, is one of the most destructive diseases of apple (Malus x domestica) worldwide, no major, qualitative gene for resistance to this disease has been identified to date in apple. We conducted a quantitative trait locus (QTL) analysis in two F(1) progenies derived from crosses between the cultivars Fiesta and either Discovery or Prima. Both progenies were inoculated in the greenhouse with the same strain of E. amylovora, and the length of necrosis was scored 7 days and 14 days after inoculation. Additive QTLs were identified using the MAPQTL: software, and digenic epistatic interactions, which are an indication of putative epistatic QTLs, were detected by two-way analyses of variance. A major QTL explaining 34.3--46.6% of the phenotypic variation was identified on linkage group (LG) 7 of Fiesta in both progenies at the same genetic position. Four minor QTLs were also identified on LGs 3, 12 and 13. In addition, several significant digenic interactions were identified in both progenies. These results confirm the complex polygenic nature of resistance to fire blight in the progenies studied and also reveal the existence of a major QTL on LG7 that is stable in two distinct genetic backgrounds. This QTL could be a valuable target in marker-assisted selection to obtain new, fire blight-resistant apple cultivars and forms a starting point for discovering the function of the genes underlying such QTLs involved in fire blight control.

[Discovery of phytopathogenic bacteria 100 years ago: transatlantic controversies and polemics]. / Découverte des bactéries phytopathogènes il y a cent ans: controverses et polémiques transatlantiques.

The demonstration of a bacterial cause of some plant diseases has been claimed few years after it was commonly recognized that bacteria were able to cause diseases of human and animal. Nevertheless, some sharp controversies took place, between German and American specialists (1897-1901), before the existence of bacterial diseases of plants was accepted by all phytopathologists. Nowadays, about 350 bacteria are described, which infect plants: they are pathovars, or subspecies, belonging to 21 genera. Bacterial diseases of plants can be classified into three major categories according to the type of symptoms shown by the infected plant: necrosis and wilt, soft-rot, tumour. The interaction between bacteria and plant cells is usually established from the apoplast, although some bacteria are xylem or phloem limited. This interaction involves an original protein secretion system (which is also described in bacteria pathogenic for animals), hydrolytic enzymes (pectinases, cellulases), toxins and/or phytohormones. Bacteria of one group (Agrobacterium) modify the plant metabolism after gene transfer from a plasmid. On the economic and social point of view, these diseases may be limiting factors of some key-productions (rice, cassava). In addition, they play a role in reducing the quality of agricultural products (reduced growth, spots on leaves and fruits). Control of bacterial diseases is limited. It relies usually on a combination of prophylaxy, chemical applications, and use of resistant genotypes.

Dual role of desferrioxamine in Erwinia amylovora pathogenicity.

To investigate the role of iron in Erwinia amylovora pathogenicity, virulence properties of two mutants of strain CFBP 1430 isolated by insertional mutagenesis and affected in the iron transport pathway mediated by desferrioxamine (DFO) were analyzed. One mutation (dfoA::MudIIpR13) disrupts DFO biosynthesis. The present analysis shows that this mutation affects an open reading frame that belongs to a biosynthetic gene cluster and shares identity with the alcA gene required for synthesis of the siderophore alcaligin in Bordetella spp. A second mutation (foxR::MudIIpR13) affects the synthesis of the ferrioxamine receptor FoxR, encoded by the foxR gene, and was shown to be transcribed into a monocistronic message. Accordingly, the foxR mutant accumulates DFO in the external medium. The growth of the mutants when supplied with various iron sources was examined; it indicates that the production of DFO and the specific transport of the DFO ferric complex are required only when iron is strongly liganded. Pathogenicity was scored after inoculation of apple seedlings and after infection of apple flowers. On seedlings, the DFO biosynthetic mutant behaved like the wild-type strain while the frequency of necrotic plants caused by the receptor mutant decreased by a factor of two to five, depending on the initial inoculum. On flowers, both mutants were strongly affected in their ability to initiate a necrotic symptom and their growth was reduced by two orders of magnitude relative to the wild-type strain. However, the virulence of the dfoA mutant varied with the inoculum concentration. Unlike the foxR mutant, the dfoA mutant only weakly induced plant cell electrolyte leakage in tobacco leaf disks. The supply with exogenous DFO, only when iron free, restored the ability to induce electrolyte leakage to the dfoA mutant and increased the leakage induced by other strains. DFO alone was not an inducer. Iron-free DFO was able to protect E. amylovora cells against lethal doses of hydrogen peroxide. The main conclusion was that production of DFO in E. amylovora during pathogenesis is not only a critical function for iron acquisition, but can play a role in the oxidative burst elicited by the bacteria.

DspA, an essential pathogenicity factor of Erwinia amylovora showing homology with AvrE of Pseudomonas syringae, is secreted via the Hrp secretion pathway in a DspB-dependent way.

In Erwinia amylovora, the dsp region, required for pathogenicity on the host plant but not for hypersensitive elicitation on tobacco, is separated from the hrp region by 4 kb. The genetic analysis reported in this paper showed that this 4kb region is not required for pathogenicity on pear seedlings. The environmental conditions allowing expression of a dsp::lacZ fusion were examined: expression was barely detected in rich medium at 30 degrees C, and the highest expression was observed in M9 galactose minimal medium at 25 degrees C. A dsp::uidA fusion appeared to be expressed only in a HrpL-proficient strain, indicating that the dsp region, like the hrp region, is positively controlled via the alternative a factor HrpL. Sequence analysis revealed that the dsp cluster encodes two genes, dspA (5517 bp) and dspB (420 bp), and that the insertions leading to the dsp::lacZ and the dsp::uidA fusions were within dspA. A HrpL-dependent promoter sequence (GGAACC-N15-CAACA) was identified upstream of dspA, and primer extension analysis detected four transcriptional starts 7, 8, 9 and 10 bp downstream of this sequence. A sigma70 promoter sequence (TTGCCC-N16-GATAAT) was observed upstream of dspB. The functionality of this second promoter was confirmed by complementation analysis. This promoter allowed constitutive expression of dspB, as measured by the expression of a dspB::uidA fusion in rich medium. In M9 galactose medium, however, HrpL was shown to activate dspB, as expression of the dspB::uidA fusion was twofold higher in a HrpL+ background than in a HrpL- background. Transposon insertions in either dspA or dspB led to a non-pathogenic phenotype. Thus, both DspA and DspB were required for E. amylovora pathogenicity, as dspB could be expressed independently of dspA. DspA and DspB were visualized as polypeptides with apparent sizes of 190 kDa and 15.5 kDa, respectively, when encoded in the T7 polymerase/promoter system. DspA, which showed homology with the protein predicted from the partial sequence of Pseudomonas syringae pv. tomato avrE transcriptional unit III, was shown to be secreted into the external medium via the Hrp secretion pathway. DspB was predicted to be acidic, like the Syc chaperone of Yersinia. A chaperone role for DspB was suggested further by the fact that DspA secretion required a functional DspB protein.

Cloning of a large gene cluster involved in Erwinia amylovora CFBP1430 virulence.

Phage MudIIPR13 insertional mutagenesis of Erwinia amylovora CFBP1430 allowed us to isolate 6900 independent CmR mutants. The frequencies of different auxotrophs in this population indicated that MudIIPR13 had inserted randomly in E. amylovora. Screening of 3500 CmR mutants on (i) apple calli and (ii) pear and apple seedlings led to the isolation of 19 non-pathogenic prototrophic single mutants, four of which expressed a LacZ+ hybrid protein. Expression of the fusion proteins was temperature sensitive. The 19 mutants could be separated into two classes according to their behaviour on tobacco: 13 were unable to elicit the hypersensitive response on tobacco (Hrp-) while six still could (Dsp-). The 19 MudIIPR13 insertions all mapped in the same virulence region. The MudIIPR13 insertions of Hrp- mutants were all clustered on the left part of this region, while the MudIIPR13 insertions of Dsp- mutants were located on the right part. All of the mutants except one, which proved to have a large deletion of the entire virulence region, could be complemented functionally by cosmids from an E. amylovora CFBP1430 genomic library. No hybridization was observed between the cosmid pPV130, which complemented 12 hrp::MudIIPR13 mutations, and the hrp genes from Pseudomonas syringae pv. phaseolicola (Lindgren et al., 1986), P. syringae pv. tomato (N.J. Panopoulos, unpublished data) or P. solanacearum (Boucher et al., 1987). Further analysis of the large virulence region will allow mapping of the border of the virulence region and facilitate the study of the function and regulation of the hrp and dsp genes.

Bacteriophage Mu as a genetic tool to study Erwinia amylovora pathogenicity and hypersensitive reaction on tobacco.

Erwinia amylovora 1430 was shown to be sensitive to Mu G(-) particles. Infection resulted either in lytic development or in lysogenic derivatives with insertion of the Mu genome at many sites in the bacterial chromosome. We used the Mu d1Bx::Tn9 (lac Apr Cmr) derivative, called Mu dX, to identify mutants affected in pathogenicity and in their ability to induce a hypersensitive reaction (HR) on tobacco plants. Inoculation of 1,400 lysogenic derivatives on apple root calli led to the identification of 12 mutants in three classes: (i) class 1 mutants were nonpathogenic and unable to induce an HR on tobacco plants; (ii) class 2 mutants were nonpathogenic but retained the ability to induce an HR; and (iii) class 3 mutants showed attenuated virulence. Of the 12 mutants, 8 had a single insertion of the Mu dX prophage. For class 1 and 2 mutants, reversion to pathogenicity was concomitant with the loss of the Mu dX prophage. Furthermore, revertants from the class 1 mutants also recovered the ability to induce an HR on tobacco plants. Five of the six class 3 mutants were impaired in exopolysaccharide production. No changes of the envelope structure (lipopolysaccharide and outer membrane proteins) were correlated with differences in pathogenicity. One class 3 mutant did not produce any functional siderophore, suggesting that iron uptake could be involved in pathogenicity.

Evidence for quantitative responses during co-culture of Pyrus communis protoplasts and Erwinia amylovora.

Mesophyll protoplasts were isolated from axenic shoot cultures of pear cultivars, exhibiting different degrees of susceptibility to fire blight infection at the whole plant level and they were co-cultured with the wild-type strain CFBP 1430 of Erwinia amylovora, and with an avirulent transposon mutant of the former (PMV 6046). Results, as assessed in terms of the effects of bacteria on protoplast viability, the time to the onset of divisions, the percentage of the originally cultivated protoplasts that divided once and of those proliferating to give 10-cell colonies, correlated with field resistance to fire blight of the respective pear genotypes. These results might provide a model for a better understanding of the interaction between pear and E. amylovora.