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  • In conclusion by investigating the

    2019-09-10

    In conclusion, by investigating the cysteine protease transcriptome in soybean nodules, we found a number of cysteine proteases belonging to the C1 and C13 families strongly up-regulated following drought exposure such as C1 cysteine protease Glyma.14G085800 and C13 cysteine proteases Glyma.17G230700 and Glyma.05G055700. However, C1 cysteine protease Glyma.14G085800 was the only protease in our study which was strongly up-regulated under drought but not up-regulated under natural senescence. By investigating an Arabidopsis α-type VPE deficient mutant we also found evidence that α-VPE Glyma.17G230700 might possibly function in C1 cysteine protease maturation, which has to be confirmed in future studies. Since there was a higher up-regulation in drought stress up-regulated cysteine protease expression compared to natural senescence, we finally found support for the view that drought induces premature senescence with accelerating tissue senescence (Muchero et al., 2009, Wehner et al., 2016). Our future work will focus on further evaluating the C1 cysteine protease Glyma.14G085800 as an early indicator for drought stress and also to silence this C1 cysteine protease to investigate the specific function of this protease under drought stress.
    Author contributions
    Funding This research was supported by the NRF National Bioinformatics and Functional Genomics program (86947) and NRF Competitive research fund (90572) ((BJV) and the NRF Incentive funding program for rated researchers (KJK). The funding received from the Genomic Research Institute, University of Pretoria, is hereby also acknowledged. MC and SGVW thank the NRF/DST and the Protein Research Foundation (MC) in South Africa for bursaries.
    Conflict of interest
    Acknowledgments
    Introduction Proteases are proteolytic enzymes that cleave the internal peptide bonds in proteins and peptides, playing important functions in all cellular organisms and being involved in a broad range of biological processes. In nematodes, proteases take part in not only in physiological processes such as embryogenesis and Manidipine 2HCl remodeling during the development of juveniles but also in parasitic processes like tissue penetration, digestion of host tissues for nutrition and evasion of host immune response (Malagón et al., 2013). In Bursaphelenchus xylophilus, the pinewood nematode (PWN), an important plant-parasitic nematode responsible for the development of the pine wilt disease and recognised as a major forest pest, the number of predicted peptidase genes is the largest in any characterised nematode genome (Kikuchi et al., 2011). Expansion of peptidases was also reflected in the secretome of B. xylophilus when compared with B. mucronatus and root-knot nematode Meloidogyne incognita secretomes (Cardoso et al., 2016, Shinya et al., 2013). Additionally, the quantitative analysis of B. xylophilus and B. mucronatus secretomes revealed a significant increase in proteases abundance in B. xylophilus (Cardoso et al., 2016). Despite both species having similar morphological and biological features, little pathogenicity to conifers has been associated with, B. mucronatus, the PWN closest related species (Kanzaki and Futai, 2006, Mamiya and Enda, 1979, Son et al., 2016). Therefore, it is expected that B. xylophilus secreted proteases play an important role in the nematode capacity to feed and parasitise pine trees causing pine wilt disease symptoms. Proteases have been divided into groups on the basis of the catalytic mechanism used during the hydrolytic process. The main catalytic types are serine, threonine, aspartate, metallo and cysteine proteases. The first cysteine protease, named papain, was purified and characterised from Carica papaya, the papaya fruit, and was also the first cysteine protease structure to be solved. Since its discovery, numerous proteases that have sequences in common with papain have been called papain-like enzymes, also named cathepsins. Papain-like, or Clan CA proteases, are further divided into families and the most common and important parasite proteases cathepsin L-like and cathepsin B belong to family C1 (Sajid and McKerrow, 2002). The main differences between these two subfamilies rely on the sequence of the pro-peptide and its length (Turk et al., 2012). The presence of cysteine proteases in B. xylophilus have been previously reported in genomic and EST (Kang et al., 2009, Kikuchi et al., 2011) studies, but no further studies on the identified cysteine proteases have been performed. According to the data in GenBank database (Benson et al., 2013), several sequences of cathepsin L-like proteins from plant-parasitic nematodes have been reported such as cathepsin L-like sequences from B. xylophilus (ACH69776; ACH56225), B. mucronatus (AID50178), Ditylenchus destructor (ACT35690), Radopholus similis (ACH56226), Rotylenchulus reniformis (AAY45870) and from different species of the genera Globodera (29 cathepsin L-like sequences), Heterodera (7) and Meloidogyne (6). However, many of these sequences are partial sequences, some of them representing just a small fragment of the complete sequence. On the other hand, cathepsin B have rarely been studied and from plant-parasitic nematodes only one sequence from B. xylophilus (ACZ13346) and other from R. similis (ADK46902) was found in GenBank.