Abstract
Pectate lyases are known to play a key role in pectin degradation by catalyzing the random cleavage of internal polymer linkages (endo-pectinases). In this paper, four novel cDNAs, designated Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7, that encode pectate lyases were cloned and characterized from the soybean cyst nematode, Heterodera glycines. The predicted protein sequences of HG-PEL-3, HG-PEL-4 and HG-PEL-6 differed significantly in both their amino acid sequences and their genomic structures from other pectate lyases of H. glycines (HG-PEL-1, HG-PEL-2 and HG-PEL-7). A phylogenetic study revealed that the pectate lyase proteins of H. glycines are clustered into distinct clades and have distinct numbers and positioning of introns, which suggests that the pectate lyase genes of H. glycines may have evolved from at least two ancestral genes. A Southern blot analysis revealed that multiple Hg-pel-6-like genes were present in the H. glycines genome. In situ hybridization showed that four novel pectate lyases (Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7) were actively transcribed in the subventral esophageal gland cells. A semi-quantitative RT-PCR assay supported the finding that the expression of these genes was strong in the egg, pre-parasitic second-stage juvenile (J2) and early parasitic J2 stages and that it declined in further developmental stages of the nematode. This expression pattern suggests that these proteins play a role in the migratory phase of the nematode life cycle. Knocking down Hg-pel-6 using in vitro RNA interference resulted in a 46.9% reduction of the number of nematodes that invaded the plants and a 61.5% suppression of the development of H. glycines females within roots compared to the GFP-dsRNA control. Plant host-derived RNAi induced the silencing of the Hg-pel-6gene, which significantly reduced the nematode infection levels at 7 Days post inoculation (dpi). Similarly, this procedure reduced the number of female adults at 40 dpi, which suggests the important roles of this gene in the early stages of parasitism. Our combined data suggest that two types of pectate lyases are present in the H. glycines genome and may have different roles during infection.
Highlights
Heterodera glycines Ichinohe, the soybean cyst nematode, is an obligatory endo-parasitic pathogen that is consistently the most damaging pest of soybean plants [1,2]
Four expressed sequence tags (ESTs) similar to the pectate lyase of H. schachtii were identified in an EST dataset of H. glycines
Four full-length cDNAs were obtained by the rapid amplification of cDNA ends (RACE)
Summary
Heterodera glycines Ichinohe, the soybean cyst nematode, is an obligatory endo-parasitic pathogen that is consistently the most damaging pest of soybean plants [1,2]. Recent estimates of the annual production losses caused by H. glycines range from $460 to $818 million in the US alone [3]. In China, the annual economic losses associated with H. glycines have been estimated to be $120 million [4]. For plant parasitic nematodes (PPNs), the cell wall, which is primarily composed of pectin and cellulose, represents a formidable barrier to penetration and migration [6,7]. The synergistic effect of several enzymes is necessary for the degradation of pectin. These enzymes can be divided into the following two main groups: pectin esterases, which remove the methoxyl groups from pectin, and depolymerases (hydrolases and lyases), which cleave the back bones among galacturonate units [8]. The pectin esterases that act on methylated pectin have not yet been recorded from PPNs [6]
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