Role of endoglucanases in Erwinia chrysanthemi 3937 virulence on Saintpaulia ionantha

The role of endoglucanases (endoglucanases Z and Y) in Erwinia chrysanthemi pathogenicity on Saintpaulia ionantha was assessed by mutagenizing cloned cel genes (celZ and celY) and recombining them with the chromosomal alleles. Strains with an omega interposon in celZ, a deletion in celY, or a double cel mutant were as virulent as the wild-type strain. However, in the strain with a deletion in celY, a delay in the appearance of symptoms was observed, and then maceration progressed as in plants infected with the wild-type strain, suggesting that E. chrysanthemi endoglucanases play a minor role in soft rot disease development.

The role of endoglucanases (endoglucanases Z and Y) in Erwinia chrysanthemi pathogenicity on Saintpaulia ionantha was assessed by mutagenizing cloned cel genes (celZ and celY) and recombining them with the chromosomal alleles. Strains with an Q interposon in celZ, a deletion in celY, or a double cel mutant were as virulent as the wild-type strain. However, in the strain with a deletion in celY, a delay in the appearance of symptoms was observed, and then maceration progressed as in plants infected with the wild-type strain, suggesting that E. chrysanthemi endoglucanases play a minor role in soft rot disease development.
Erwinia chrysanthemi 3937, a soft rot disease plant bacterium, produces and secretes several depolymerizing enzymes, including pectinases, endoglucanases (EGs), and proteases. The major pectate lyases of E. chrysanthemi 3937 and pectin methylesterase have been shown to be necessary for full pathogenicity on Saintpaulia ionantha (5,6). However, a mutant of E. chrysanthemi 3937 with its five pectate lyase activities deleted has been shown to be noninvasive but was still able to macerate the inoculated leaves of Saintpaulia plants and to produce a limited maceration on potato tubers (1, 3), suggesting a putative role for other depolymerizing enzymes produced by E. chrysanthemi.
The role of EG in the pathogenicity of Pseudomonas solanacearum and Xanthomonas campestris has been studied (11,18). The virulence of an EG-deficient strain depended on the bacterial species. A delay in the appearance of symptoms and killing of tomato plants was observed in an EG-deficient strain of P. solanacearum. However, an X campestris mutant lacking EG showed little reduction in virulence. E. chrysanthemi 3937 produces two EGs: EGZ, which comprises the major activity and is secreted (8), and EGY, which is less abundant and is periplasmic (12). The genes encoding these activities, celZ and celY, respectively, have been mapped in two different locations of the chromosome, and the conditions of their optimum expression have been studied with lacZ fusions (2). To study the contribution of E. chrysanthemi EGs in soft rot disease, we chose not to use the previously described cel mutants, which were lacZ translational fusions (2), but instead constructed new cellY and celZ mutants and analyzed their pathogenicity on Saintpaulia plants.
A celZ mutant was constructed as follows. The fQ interposon encoding resistance to spectinomycin and streptomycin and carrying transcriptional and translational termination signals was obtained after SmaI digestion of the pHP45fQ plasmid (15). This SmaI fragment was inserted into the Klenow fragment-filled ClaI site of the celZ-carrying plasmid pAJ24 (13) (Fig. 1). The mutated plasmid was introduced into strain 3937 (wild type), and after marker exchange (19), the E-1006 strain was selected. described by Ried and Collmer (17). A PstI fragment carrying the sac genes of Bacillus subtilis and the npt gene of Escherichia coli was obtained through partial digestion of the pUM24 plasmid and was introduced into the PstI site of the celYcontaining plasmid pMH17 (7). Exchange recombination between the mutated cloned celY gene and the chromosomal allele was then selected. Spontaneous excision of the npt-sacB-sacR fragment was then selected in the presence of 1% sucrose. To measure the extent of the deletion, strain E-1008 DNA was hydrolyzed with BamHI, SmaI, or PstI. After transfer, the DNA blot was hybridized with the BamHI-SmaI fragment from pMH17 (Fig. 1). Strain E-1008, which had lost the SmaI site and suffered a deletion of 2 kb overlapping the celY gene, was retained (data not shown).
The E-1006 and E-1008 mutant strains were checked by electrofocusing and were found to be negative for EGZ and EGY (Fig. 2), respectively, before their inoculation into Saintpaulia plants. To construct a double celY celZ mutant (E-1014), the cel::fl mutation carried by the E-1006 strain was introduced into the E-1008 strain by transduction with the phage 4)EC2 (16). No EG was produced by strain E-1014 (Fig.   2).
When strain 3937 was inoculated into plants, a translucent spot at the inoculation site was visible after 24 h. After 3 days, the maceration had affected all of the leaf blade and sometimes had affected the petiole. A week later, maceration had propagated to the whole leaf (Fig. 3A), and 2 weeks later, the whole plant was diseased (70% systemic responses). When the E-1006 mutant was inoculated on Saintpaulia plants, the progression of symptoms of E-1006 infection over a week was identical to that of the wild-type strain. The progression of symptoms was not as rapid when strain E-1008 was inoculated (Fig. 3B). However, a week after inoculation, no difference in the number of diseased plants and the severity of symptoms between the wild type, cel mutants, and the double cel mutant (P < 0.05 [ Fig. 4]) was observed.
The mutant E-1006, which is depleted of E. chrysanthemi major EG (EGZ), is as virulent as the wild-type strain. In the E-1008 mutant, a slight delay (3 days) in the progression of symptoms was observed, suggesting that EGY is necessary in the early stages of infection. In the infected plant, the bacteria develop in the intercellular space and are in contact with the pectin-rich middle lamella. The delay observed with E-1008 is surprising, because EGY is a minor and periplasmic EG. that the substrate specificity of EGY is broader than that of EGZ (2a, 9). This could suggest that EGY hydrolyzes other substrates present in the middle lamella, making the pectin more accessible to pectinases. However, as the level of pectate lyase increases (14), EGY is no longer required, and the maceration progresses as in the wild-type strain. Our results suggest that the EGs of E. chrysanthemi 3937 are not essential for soft rot disease development on Saintpaulia plants. Thus, the role of the EG might be limited to the saprophytic life of E. chrysanthemi; on dead tissues cellulose already attacked by fungi might be a better substrate for bacterial EGs, providing nutriments for the bacteria.  . Distribution of responses induced by wild-type and mutant strains on Saintpaulia plants. Symptoms produced after 1 month were recorded. The symptoms were classified into three groups: *, systemic response (the whole plant is macerated); E, localized response (maceration limited to the inoculated leaf); and U, local necrosis or no symptoms. The results are averages of three to five experiments; each experiment was performed with 20 plants. Standard error bars are indicated. Analysis of variance demonstrated that there were no significant differences between the means of the three groups (P < 0.05).
We thank our colleagues R. Vedel for technical tips, J. Laurent for statistical expertise, and D. Expert for support during this work.