Engineered resistance

1.2.9.3.1 Pathogen derived resistance

The expression of viral CPs within potato plants (CP-mediated resistance) was the first successful approach in molecular biological potato virus resistance research (Abel et al. 1986). In the beginning of the 90s, the varieties ˈBintjeˈ, ˈEscortˈ and ˈRusset Burbankˈ were transformed in order to express the coat proteins of PVX (Hoekema et al. 1989; Kaniewski et al. 1990), PVY (Kaniewski et al. 1990) and PLRV (van der Wilk et al. 1991; Kawchuk et al. 1991) leading to improved resistance against infection by these viruses. CP-mediated resistance comprises the insertion of sense and/or antisense sequences of viral structural proteins, in this case the coat protein (Waterhouse et al. 1998). The mechanism behind, is not yet fully understood (Bendahmane et al. 2007). Mode of action theories are the inhibition of viral RNA translation by viral target protein accumulation or accumulation of the viral RNA itself, inhibiting viral multiplication in a dose-dependent manner (Besong-Ndika et al. 2015; Galvez et al. 2014) or a RNA-mediated mechanism leading to RNA silencing processes (Waterhouse et al. 1998; Zerbini et al. 2005; Galvez et al. 2014). Other genomic sequences used in that sort of resistance are coding for viral non-structural proteins like replicase proteins or movement proteins, which are thought to act in a RNA-mediated manner, as well (Mueller et al. 1995; Galvez et al. 2014). In that respect, RNA silencing is due to sequence identity between the transgenic plant derived RNA and the viral RNA leading to dsRNA formation. The created dsRNAs serve as substrate for the RNaseIII-like enzyme Dicer, which cleaves the long dsRNA into siRNAs. Incorporation of siRNAs into the RISC complex leads to viral RNA silencing (Novina and Sharp 2004). However, viral transgenes transformed as sense or anti-sense are often unstable and regularly yield in partial resistance (Mäki-Valkama et al. 2000; Arif et al. 2012). Broad-spectrum resistance approaches (Ai et al. 2011; Arif et al. 2012; Hameed et al. 2017) and lower viral recombination probability (Tenllado et al. 2004) was achieved by using short viral sequences, like hairpin RNAs and modified hairpin RNAs, called artificial microRNAs (amiRNAs; Song et al. 2014; Ai et al. 2011) instead of using the whole viral sequence coding for one protein to produce siRNAs. For stimulation of dsRNA formation and therefore stimulation of sequence specific RNA silencing machinery prior to virus infection, inverted repeat constructs of sense and antisense viral genome sequences were designed and contributed to higher resistance stability (Hameed et al. 2017; Prins et al. 2008; Waterhouse et al. 1998). In theory, viral RNA is promptly targeted and degraded even before virus-encoded

infection (Waterhouse and Helliwell 2003), as well as against viruses with related genomic sequence up to 93% sequence similarity (Missiou et al. 2004). The silencing efficiency in pathogen derived engineered resistance is highly depending on the applied sequences in combination with incorporation affinity of the RISC complex (Chen et al. 2010; Jiang et al. 2011; Song et al. 2014).

Nevertheless, the probability of viral recombination events in plants carrying pathogen derived resistance infected with avirulent viruses was proofed in the laboratory under high selective pressure. Different research groups demonstrated virulence increase under selective conditions (Schoelz and Wintermantel 1993; Greene and Allison 1994; Aaziz and Tepfer 1999; Tepfer et al. 2015). However, Flatken (2006) and Dietrich et al. (2007) compared recombination frequency of several potato viruses in transgenic and non-transgenic potato plants. They found a low emergence likelihood of novel viruses with virulent prospective (Dietrich et al. 2007; Tepfer et al. 2015). In pathogen derived resistance, short viral sequences should be favored in order to lower recombination probability (Aaziz and Tepfer 1999; Tenllado et al. 2004).

1.2.9.3.2 Pathogen targeted resistance

Compared to pathogen derived resistance described before, the expression of non-viral sequences in pathogen targeted resistance transgenic plants probably lowers the viral recombination risk (Aaziz and Tepfer 1999; Gargouri-Bouzid et al. 2006). The generation of artificial “plantibodies”, binding on functional domains of viral proteins, lead to virus inactivation and therefore to prevention of viral infestation (Safarnejad et al. 2011). For example, cytosolic (Ayadi et al. 2012), as well as apoplastic (Gargouri-Bouzid et al. 2006) expression of single- chain variable Fragment (scFv) antibodies targeting the protease Nla of PVY, resulted in transgenic plants displaying efficient inhibition of virus multiplication. Additionally, Nickel et al. (2008) reduced PLRV accumulation by scFv antibody-mediated inhibition targeting the P1 protein. Recently, the application of 3D8 scFv antibodies with nucleic acid hydrolyzing activity provoked broad-spectrum resistance to DNA and RNA genomic viruses in tobacco and potato plants (Yang et al. 2017). Yang et al. (2017) challenged these transgenic plants with PVX and discovered PVX-tolerance referring to the expression of 3D8 scFv antibodies.

Targeted genome editing (TGE) techniques generating transgenic and cisgenic plants are specific and directable (Hartung and Schiemann 2014; Small and Puchta 2014; van Eck 2018). In contrast to transgenic crops, cisgenic plants only exhibit genes deriving from other varieties or relatives (Hou et al. 2014). These single genes could naturally be transferred by sexual hybridization or by using conventional breeding techniques and allows cultivar improvement with only innate alleles, including resistance alleles, from the breeders gene pool in a shorter time (Hou et al. 2014; Schouten et al. 2006). Cis-plant genomic modifications achieved by TGE

Introduction

technologies are suggested to be indistinguishable from natural mutations in the conventional counterparts (Jacobsen and Schouten 2009; Davison and Ammann 2017; Ishii and Araki 2017). Since the last decade there is discussion about distinguishing cisgenic plants from transgenic plants regarding genetically modified organisms (GMOs) regulations for liberation and cultivation (Schouten et al. 2006; Ishii and Araki, 2017; van Eck 2018).

TGE techniques employ engineered nucleases, including meganucleases (Epinat et al. 2003; Osakabe and Osakabe 2015), zinc finger nucleases (ZFNs; Kim et al. 1996; Osakabe and Osakabe 2015), transcription activator-like effector nucleases (TALENs; Bogdanove and Voytas 2011; Osakabe and Osakabe 2015), and clustered regularly interspaced short palindromic repeats /associated protein nucleases (CRISPR/Cas; Jinek et al. 2012; Doudna and Charpentier 2014; Subburaj et al. 2016) to mediate specific nucleic acid sequence editing. To facilitate TGE, delivery methods of TGE reagents are needed and denoted as transformation methods, this also includes protoplasts (Andersson et al. 2017), particle bombardment and Agrobacterium based mediation (Wang et al. 2015; Ma et al. 2017).

In potato, the modification of the acetolacetate synthase gene (Nicolia et al. 2015), improved cold storage and processing traits (Clasen et al. 2016) and modification of starch branching enzyme and an acid invertase (Ma et al. 2017) were achieved by TALENs. CRISPR/Cas was used for further alteration studies on the acetolacetate synthase gene (Butler et al. 2015), to produce auxin/indole-3-acetic acid gene knock out potato plants (Wang et al. 2015) and to modify starch quality (Andersson et al. 2017). Till now, we do not know of any cisgenic food crop potato plants with potato virus resistance genes from Solanum section Petota species achieved by TGE techniques (Malzahn et al. 2017; van Eck 2018).