Cloning and characterization of a pectin lyase gene from Colletotrichum lindemuthianumand comparative phylogenetic/structural analyses with genes from phytopathogenic and saprophytic/opportunistic microorganisms

Abstract

BackgroundMicroorganisms produce cell-wall-degrading enzymes as part of their strategies for plant invasion/nutrition. Among these, pectin lyases (PNLs) catalyze the depolymerization of esterified pectin by a β-elimination mechanism. PNLs are grouped together with pectate lyases (PL) in Family 1 of the polysaccharide lyases, as they share a conserved structure in a parallel β-helix. The best-characterized fungal pectin lyases are obtained from saprophytic/opportunistic fungi in the genera Aspergillus and Penicillium and from some pathogens such as Colletotrichum gloeosporioides.The organism used in the present study, Colletotrichum lindemuthianum, is a phytopathogenic fungus that can be subdivided into different physiological races with different capacities to infect its host, Phaseolus vulgaris. These include the non-pathogenic and pathogenic strains known as races 0 and 1472, respectively.ResultsHere we report the isolation and sequence analysis of the Clpnl2 gene, which encodes the pectin lyase 2 of C. lindemuthianum, and its expression in pathogenic and non-pathogenic races of C. lindemuthianum grown on different carbon sources. In addition, we performed a phylogenetic analysis of the deduced amino acid sequence of Clpnl2 based on reported sequences of PNLs from other sources and compared the three-dimensional structure of Clpnl2, as predicted by homology modeling, with those of other organisms. Both analyses revealed an early separation of bacterial pectin lyases from those found in fungi and oomycetes. Furthermore, two groups could be distinguished among the enzymes from fungi and oomycetes: one comprising enzymes from mostly saprophytic/opportunistic fungi and the other formed mainly by enzymes from pathogenic fungi and oomycetes. Clpnl2 was found in the latter group and was grouped together with the pectin lyase from C. gloeosporioides.ConclusionsThe Clpnl2 gene of C. lindemuthianum shares the characteristic elements of genes coding for pectin lyases. A time-course analysis revealed significant differences between the two fungal races in terms of the expression of Clpnl2 encoding for pectin lyase 2. According to the results, pectin lyases from bacteria and fungi separated early during evolution. Likewise, the enzymes from fungi and oomycetes diverged in accordance with their differing lifestyles. It is possible that the diversity and nature of the assimilatory carbon substrates processed by these organisms played a determinant role in this phenomenon.

Cloning and characterization of an endo--1,4-xylanase gene from Colletotrichum lindemuthianum and phylogenetic analysis of similar genes from phytopathogenic fungus

“…Additionally, the pathogenic race degrades xylan faster and grows better than the non-pathogenic race, suggesting a different ability in the degradation of this polysaccharide and the use of oligo-or monomeric sugars. As previously described (Hernández-Silva et al, 2007;Lara-Marquez et al, 2011), the expression of Clpnl2 gene and activity of pectin lyase between the two races were similar to that observed in this study when 92% esterified pectin was utilized as the sole carbon source. In other words, the pathogen requires a rapid and higher level expression of endoxylanase activity and other related lyticases for successful interaction with the live plant tissue, which implies an energy cost and the non-pathogen does cannot invest because it feeds on dead plant tissue.…”

“…Race 0 does not seem to prefer xylan as a carbon source but instead grows better with bean cell walls, suggesting that differences exist in the utilization of mono-or oligosaccharides on race 1472. The authors observed a similar behavior of other enzymes of the complex involved in the degradation of the cell wall suggesting that it may be a general phenomenon (Acosta-Rodriguez et al, 2005;Hernández-Silva et al, 2007;Lara-Marquez et al, 2011). The differences at this level can be part of the general response of fungi to host components.…”

Evolution and functional characterization of pectate lyase PEL12, a member of a highly expanded Clonostachys rosea polysaccharide lyase 1 family

“…Analysis and hierarchical clustering of gene numbers in a set of different fungal genomes revealed a clear distinction of PL1 gene distribution related to nutritional mode; Trichoderma mycoparasites and entomopathogenic fungi have none or drastically reduced PL1 gene numbers, while PL1 gene numbers in plant pathogenic fungi such as N. haematococca , Colletotrichum and Verticillium are highly expanded. Plant pathogenic fungi use plant cell wall-degrading enzymes for penetration and nutrient acquisition, while plants have evolved strategies that allow them to detect and to defend against the attack of pathogens by producing inhibitors of these enzymes [ 22 ]. Pectic structures are also extremely diverse, depending on the plant and plant tissue.…”

“…Our phylogenetic analysis of the PL1 gene family does not contradict this hypothesis, as we detected a high number of hierarchically organized subgroups and divisions, including two previously unknown PL1 subgroups, which may represent isozymes with particular properties. For example, significant differences between two fungal races of C. lindemuthianum were detected in terms of the expression of the Clpnl2 gene encoding for pectin lyase 2, where the pathogenic race 1472 responded faster and with higher expression levels than the non-pathogenic race 0 [ 22 ]. Wijesundra et al [ 39 ] reported that C. lindemuthianum race β secreted two forms of pectin lyase, having pI values of 8.2 and 9.7, respectively, when grown in culture with sodium polypectate or isolated Phaseolus vulgaris hypocotyl cell walls as the main carbon source.…”

“…Several fungal pectin/pectate lyases from a variety of microbial species have been functionally characterized, such as the saprotrophic/opportunistic Aspergillus niger [ 13 – 16 ], A. oryzae [ 16 , 17 ], Penicillium griseoroseum [ 18 ], P. occitanis [ 19 ], and the phytopathogenic fungi Glomerella cingulata [ 20 ] Colletotrichium gloeosporioides [ 21 ], C . lindemuthianum [ 22 ] and Botrytis cinerea [ 23 ]. Recently, comparative genome analysis of Clonostachys rosea (Hypocreales, Bionectriaceae) revealed that PL1 is one of the most significantly expanded gene families in this ubiquitous mycoparasitic fungus compared to nine closely related Sordariomycetes [ 24 ].…”