Inadvertent gene silencing of argininosuccinate synthase (bcass1) in Botrytis cinerea by the pLOB1 vector system

Abstract

For several years, researchers working on the plant pathogen Botrytis cinerea and a number of other related fungi have routinely used the pLOB1 vector system, based on hygromycin resistance, under the control of the Aspergillus nidulans oliC promoter and what was reported to be the beta-tubulin (tubA) terminator. Recently, it has been demonstrated that this vector contains a 446-bp portion of the B. cinerea argininosuccinate synthase gene (bcass1) rather than the tubA terminator. As argininosuccinate synthase is essential for the production of L-arginine, inadvertent gene silencing of bcass1 may result in partial L-arginine auxotrophy and, indeed, may lead to altered phenotypes in planta. In this article, we report our findings relating to possible problems arising from this incorrect plasmid construction. As an absolute baseline, gene disruption of bcass1 was carried out and generated a strict auxotroph, unable to grow without exogenous arginine supplementation. The knockout displayed an alteration in host range in planta, showing a reduction in pathogenicity on strawberries, French bean leaves and tomatoes, but maintained wild-type growth on grape, which is in accordance with the reported arginine availability in such tissues. Deliberate gene silencing of bcass1 mirrored these effects, with strongly silenced lines showing reduced virulence. The degree of silencing as seen by partial auxotrophy was correlated with an observed reduction in virulence. We also showed that inadvertent silencing of bcass1 is possible when using the pLOB1 vector or derivatives thereof. Partial arginine auxotrophy and concomitant reductions in virulence were triggered in approximately 6% of transformants obtained when expressing enhanced green fluorescent protein, luciferase, monomeric red fluorescent protein or beta-glucuronidase using the pLOB1-based expression system, which inadvertently contains 446 bp of the bcass1 coding sequence. We recommend the testing of transformants obtained using this vector system for arginine auxotrophy in order to provide assurance that any observed effects on the development or virulence are a result of the desired genetic alteration rather than accidental bcass1 silencing.

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“…The genome sequences of two B. cinerea strains failed to identify 'silver bullets', unique features which distinguish it from other pathogenic and nonpathogenic fungi (Amselem et al, 2011). The availability of the genome sequence and a variety of molecular tools [ease of transformation to obtain knockout mutants (van Kan et al, 1997) or to achieve gene silencing (Patel et al, 2008(Patel et al, , 2010], together with its economic relevance, have contributed to B. cinerea being the most extensively studied necrotrophic fungal pathogen. These studies have considerably advanced our understanding of the infection strategies of B. cinerea, yet only very few absolutely essential virulence determinants have been identified by candidate gene approaches (Tudzynski and Kokkelink, 2009).…”

MoARG1, MoARG5,6 and MoARG7 involved in arginine biosynthesis are essential for growth, conidiogenesis, sexual reproduction, and pathogenicity in Magnaporthe oryzae

“…Previously, numerous studies have demonstrated that amino acid metabolism plays an important role in the growth and virulence in many pathogenic fungi (Namiki et al, 2001;Seong et al, 2005;Gong et al, 2007;Patel et al, 2010;Takahara et al, 2012). In M. oryzae, two REMI transformants pth3 and met1 have been identified, which exhibit histidine auxotrophy and methionine auxotrophy, respectively (Sweigard et al, 1998;Balhadère et al, 1999).…”

Cys 2 His 2 Zinc Finger Transcription Factor BcabaR1 Positively Regulates Abscisic Acid Production in Botrytis cinerea

“…In the filamentous fungus, RNA-mediated gene silencing is a reliable genetic manipulation strategy in analyzing gene function (29). Patel et al (30) first reported RNA silencing of a superoxide dismutase gene in B. cinerea, and the functions of several other B. cinerea genes that participate in pathogenicity or other biological processes have since been investigated using RNA interference (31)(32)(33). We used a hairpin RNA (hpRNA) transgene-based gene knockdown strategy to successfully develop RNA interference in B. cinerea and found partial silencing in all positive transformants.…”