Expression of serine proteases in egg-parasitic nematophagous fungi during barley root colonization

Abstract

a b s t r a c tNematophagous fungi Pochonia chlamydosporia and P. rubescens colonize endophytically barley roots. During nematode infection, serine proteases are secreted. We have investigated whether such proteases are also produced during root colonization. Polyclonal antibodies against serine protease P32 of P. rubescens cross-reacted with a related protease (VCP1) of P. chlamydosporia, but not with barley proteases. These antibodies also detected an unknown ca. 65-kDa protein, labeled hyphae and appressoria of P. chlamydosporia and strongly reduced proteolytic activity of extracts from fungus-colonized roots. Mass spectrometry (MS) of 32-kDa protein bands detected peptides homologous to VCP1 only in Pochonia-colonized roots. Peptides homologous to barley serine carboxypeptidases were found in 65 kDa bands of all roots. RT-PCR detected expression of VCP1 and a new P. chlamydosporia serine carboxypeptidase (SCP1) genes only in fungus-colonized roots. SCP1 shared limited sequence homology with VCP1 and P32. Expression in roots of proteases from nematophagous fungi could be greatly relevant for nematode biocontrol.

Sequencing and functional analysis of the genome of a nematode egg-parasitic fungus, Pochonia chlamydosporia

“…To this respect, the nematode Meloidogyne incognita genome contains genes related to the immune response and antifungal defenses (Abad et al, 2008). Expression of the VCP1 protease and the serine carboxypeptidase SCP1 has been detected during P. chlamydosporia endophytism (Lopez-Llorca et al, 2010). Additionally, we have identified in this work several families of expressed proteases putatively related to fungal-plant interaction, such as the rhomboid protease family (S54), likely involved in its endophytic behavior.…”

“…5A). Among these enzymes, subtilisins and serine carboxypeptidases are known to be involved in nematode egg-parasitism (LopezLlorca et al, 2002b;Ward et al, 2011) and expressed during root endophytic colonization (Lopez-Llorca et al, 2010) by this fungus. We found in this context that the P. chlamydosporia genome coded for 32 serine proteases of the S8 family (subtilisins) and 16 proteases of the S10 family (serine carboxypeptidases), 7 of the latter being putatively secreted enzymes (Fig.…”

“…root papillae formation) plant defenses (Bordallo et al, 2002;Escudero and Lopez-Llorca, 2012;Macia-Vicente et al, 2009a), but these do not prevent root colonization by the fungus. P. chlamydosporia expresses proteases during this process, such as VCP1 and SCP1, the latter a newly reported serine carboxypeptidase (Larriba et al, 2012;Lopez-Llorca et al, 2010).…”

Proteome of the Nematode-Trapping Cells of the Fungus Monacrosporium haptotylum

“…In addition to subtilisins, two carboxypeptidases were upregulated in knobs. One of them (H072_11104) shows sequence similarity to a carboxypeptidase that is produced during root colonization by the egg-parasitic nematophagous fungus Pochonia chlamydosporia (65). The other upregulated carboxypeptidase (H072_4030) shows sequence similarity to carboxypeptidase Y in M. grisea, which is expressed during appressorium formation (66).…”

Microbial Protection Against Plant Disease

“…For example, bacterial and fungal chitinases have been shown to degrade fungal cell walls and degrade chitinous components of nematode eggs (Cohen-Kupiec and Chet 1998; Neeraja et al 2010); proteases produced by biocontrol bacteria and fungi have been shown to be involved in antifungal activity and nematode predation (Ko et al 2009;Lopez-Llorca et al 2010). Chitin monomers produced by BCA-mediated chitinase activity can also serve to elicit ISR in plants (see below), indicating that in certain cases BCAs can inhibit pathogens by more than one antagonistic mechanism.…”