The Pochonia chlamydosporia Serine Protease Gene vcp1 Is Subject to Regulation by Carbon, Nitrogen and pH: Implications for Nematode Biocontrol

The alkaline serine protease VCP1 of the fungus Pochonia chlamydosporia belongs to a family of subtilisin-like enzymes that are involved in infection of nematode and insect hosts. It is involved early in the infection process, removing the outer proteinaceous vitelline membrane of nematode eggs. Little is known about the regulation of this gene, even though an understanding of how nutrients and other factors affect its expression is critical for ensuring its efficacy as a biocontrol agent. This paper provides new information on the regulation of vcp1 expression. Sequence analysis of the upstream regulatory region of this gene in 30 isolates revealed that it was highly conserved and contained sequence motifs characteristic of genes that are subject to carbon, nitrogen and pH-regulation. Expression studies, monitoring enzyme activity and mRNA, confirmed that these factors affect VCP1 production. As expected, glucose reduced VCP1 expression and for a few hours so did ammonium chloride. Surprisingly, however, by 24 h VCP1 levels were increased in the presence of ammonium chloride for most isolates. Ambient pH also regulated VCP1 expression, with most isolates producing more VCP1 under alkaline conditions. There were some differences in the response of one isolate with a distinctive upstream sequence including a variant regulatory-motif profile. Cryo-scanning electron microscopy studies indicated that the presence of nematode eggs stimulates VCP1 production by P. chlamydosporia, but only where the two are in close contact. Overall, the results indicate that readily-metabolisable carbon sources and unfavourable pH in the rhizosphere/egg-mass environment may compromise nematode parasitism by P. chlamydosporia. However, contrary to previous indications using other nematophagous and entomopathogenic fungi, ammonium nitrate (e.g. from fertilizers) may enhance biocontrol potential in some circumstances

Pratylenchidae – the lesion nematodes

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Morphological and molecular characterisation of Helicotylenchus pseudorobustus (Steiner, 1914) Golden, 1956 and related species (Tylenchida: Hoplolaimidae) with a phylogeny of the genus

Morphological identification of spiral nematodes of the genus Helicotylenchus is a difficult task because most characters used for their diagnosis vary within species. In this paper we provide morphological and molecular characterisations of several spiral nematodes, H. broadbalkiensis, H. digonicus, H. dihystera, H. microlobus, H. paxilli and H. pseudorobustus, collected in different geographical areas of USA, Switzerland, Italy, New Zealand, Spain, UK, South Korea and Russia. We suggest that H. microlobus and H. pseudorobustus are valid species separated from each other morphologically and molecularly. Seven species with distinct molecular characteristics are also distinguished, but are not ascribed morphologically to any specific taxon because of the low number of specimens available. Phylogenetic relationships of H. pseudorobustus with other Helicotylenchus species are given as inferred from the analyses of 154 sequences of the D2-D3 of 28S rRNA gene and 37 sequences of ITS rRNA gene.This research was partially supported by grant AGL2012-37521 from ‘Ministerio de Economía y Competitividad’ of Spain, grant P12-AGR 1486 and grant AGR-136 from ‘Consejería de Economía, Innvovación y Ciencia’ from Junta de Andalucía and Union Europea, Fondo Europeo de Desarrollo regional, ‘Una manera de hacer Europa’, and the Russian Foundation of Basic Research, project number 14-04-00953.Peer reviewe

<i>Pochonia chlamydosporia</i> isolates used.

1<p>The source location indicates a root-knot nematode host <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035657#pone.0035657-Bourne2" target="_blank">[61]</a>.</p>2<p>A P. <i>chlamydosporia</i> var. <i>catenulata</i> strain.</p

The effect of glucose and <i>M. incognita</i> eggs on VCP1 enzyme and mRNA production.

<p>Different isolates of <i>P. chlamydosporia</i> were grown in 0.01M potassium phosphate buffer pH 6.8 (P) with or without 2% glucose (G) and eggs (E, 1egg/µl). The effect of eggs was not tested on Pc147 and Pc392. Error bars denote standard errors of 3 biological replicates. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035657#pone-0035657-g002" target="_blank">Fig. 2</a>a, * denotes where the P+G and P+G+E means were significantly different (p<0.05) from the corresponding P mean; in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035657#pone-0035657-g002" target="_blank">Fig. 2</a>b all of the P+G and P+G+E means were significantly different from their corresponding P mean (p<0.05). In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035657#pone-0035657-g002" target="_blank">Fig. 2</a>b, † indicates where the P+G+E mean was significantly different (p<0.05) from the P+G mean. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035657#pone-0035657-g002" target="_blank">Fig. 2</a>a none of the P+G+E means was significantly different from the corresponding P+G mean.</p

The effect of pH on VCP1 enzyme and mRNA production.

<p>Different isolates of <i>P. chlamydosporia</i> were grown in 0.01M potassium phosphate buffers at different pHs (pH 5.8, pH 6.8 and pH 8). Error bars denote standard errors of the biological replicates. An asterisk denotes that the mean value is significantly different (p<0.05) from the corresponding pH 6.8 mean and † denotes that the mean value is significantly different (p<0.05) from the corresponding pH 5.8 mean.</p

Positions of putative regulatory motifs upstream of the VCP1 coding region in <i>P. chlamydosporia</i> isolates.

<p>The sequences of these motifs (5′ to 3′) are as follows: CREA = SYGGRG; CREB = CAGCGTCA; GATA = GATA; PacC = GCCARG, where S = C/G, Y = C/T and R = G/A. Positions given correspond to the 5′ end of the motif except where the motif is on the complementary strand (*) when the 3′end is shown. Distance upstream indicates the distance from the transcript start. Y indicates the sequence conforms to the consensus, N that it does not.</p

Cryo-SEM micrographs of Pc10 incubated with <i>M. incognita</i> eggs for 24h in different media.

<p>Isolate Pc10 was incubated with eggs for 24 h in phosphate buffer (P, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035657#pone-0035657-g005" target="_blank">Fig. 5</a>A-B), P+2% glucose (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035657#pone-0035657-g005" target="_blank">Fig. 5</a>C–D) or P+ 200 mM ammonium chloride (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035657#pone-0035657-g005" target="_blank">Fig. 5</a>E–H). In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035657#pone-0035657-g005" target="_blank">Fig. 5</a>A, B, C, E and G, bar = 10 µm, in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035657#pone-0035657-g005" target="_blank">Fig. 5</a>D, E inset, F and H, bar = 1 µm.</p