Cloning, disruption, and expression of two endo-beta 1, 4-xylanase genes, XYL2 and XYL3, from Cochliobolus carbonum

Abstract

In culture, the filamentous fungus Cochliobolus carbonum, a pathogen of maize, makes three cationic xylanases. XYL1, which encodes the major endoxylanase (Xyl1), was earlier cloned and shown by gene disruption to encode the first and second peaks of xylanase activity (P. C. Apel, D. G. Panaccione, F. R. Holden, and J. D. Walton, Mol. Plant-Microbe Interact. 6:467-473, 1993). Two additional xylanase genes, XYL2 and XYL3, have now been cloned from C. carbonum. XYL2 and XYL3 are predicted to encode 22-kDa family G xylanases similar to Xyl1. Xyl2 and Xyl3 are 60% and 42% identical, respectively, to Xyl1, and Xyl2 and Xyl3 are 39% identical. XYL1 and XYL2 but not XYL3 mRNAs are present in C. carbonum grown in culture, and XYL1 and XYL3 but not XYL2 mRNAs are present in infected plants. Transformation-mediated gene disruption was used to construct strains mutated in XYL1, XYL2, and XYL3. Xyl1 accounts for most of the total xylanase activity in culture, and disruption of XYL2 or XYL3 does not result in the further loss of any xylanase activity. In particular, the third peak of cationic xylanase activity is still present in a xyl1 xyl2 xyl3 triple mutant, and therefore this xylanase must be encoded by yet a fourth xylanase gene. A minor protein of 22 kDa that can be detected immunologically in the xyl1 mutant disappears in the xyl2 mutant and is therefore proposed to be the product of XYL2. The single xylanase mutants were crossed with each other to obtain multiple xylanase disruptions within the same strain. Strains disrupted in combinations of two and in all three xylanases were obtained. The triple mutant grows at the same rate as the wild type on xylan and on maize cell walls. The triple mutant is still fully pathogenic on maize with regard to lesion size, morphology, and rate of lesion development.

Simultaneous silencing of endo‐β‐1,4 xylanase genes reveals their roles in the virulence of Magnaporthe oryzae

“…In contrast to this study, most of the previous efforts with disruption mutants of single or even multiple endoxylanase genes failed to show a positive contribution of the enzymes to the pathogenicity of fungal plant pathogens (Apel-Birkhold and Walton, 1996;Keen et al, 1996;Wu et al, 1997). This can be explained by endoxylanases as a group and not specific endoxylanase genes being important for fungal pathogenesis.…”

Correlation of in planta endo‐beta‐1,4‐xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola

“…Cloning of the putative xylanase genes involved here should pave the way for complementary approaches such as gene disruption techniques to determine specific roles of xylanases during pathogenesis by M. graminicola. However, targeted disruption of individual xylanase genes from C. carbonum (Apel et al, 1993;Apel-Birkhold & Walton, 1996) did not dramatically affect pathogenicity. Functional redundancy or the occurrence of multiple xylanase genes has been put forward as one of the main reasons why xylanase gene-disruption mutants remain pathogenic (Apel-Birkhold & Walton, 1996).…”

“…However, targeted disruption of individual xylanase genes from C. carbonum (Apel et al, 1993;Apel-Birkhold & Walton, 1996) did not dramatically affect pathogenicity. Functional redundancy or the occurrence of multiple xylanase genes has been put forward as one of the main reasons why xylanase gene-disruption mutants remain pathogenic (Apel-Birkhold & Walton, 1996). An alternative approach to determine the role of multiple xylanase genes in pathogenesis is the disruption of transcriptional regulators or conserved signal transduction components, since these proteins normally affect the whole set of xylanase genes (Beliën et al, 2006).…”

“…Xylanase genes have been cloned in wide range of plant pathogenic fungi, such as Cochliobolus carbonum (Apel et al, 1993;Apel-Birkhold & Walton, 1996) and Botrytis cinerea (Brito et al, 2006). Cloning of the putative xylanase genes involved here should pave the way for complementary approaches such as gene disruption techniques to determine specific roles of xylanases during pathogenesis by M. graminicola.…”

Identification of a putative voltage‐gated Ca2+ channel as a key regulator of elicitor‐induced hypersensitive cell death and mitogen‐activated protein kinase activation in rice

“…, 1998). Xylanases have been isolated and characterized from a variety of different plant pathogenic fungi, including rice blast fungus (Apel‐Birkhold and Walton, 1996; Giesbert et al. , 1998; Luttig et al.…”