Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications

Biotechnology Advances 33 (2015) 1024–1042

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Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications Pavel Krenek, Olga Samajova, Ivan Luptovciak, Anna Doskocilova, George Komis, Jozef Samaj ⁎ Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Cell Biology, Faculty of Science, Palacký University, Šlechtitelů 27, CZ-783 71 Olomouc, Czech Republic

a r t i c l e

i n f o

Available online 25 March 2015 Keywords: Agrobacterium tumefaciens Virulence Transient transformation RNA interference Virus induced gene silencing Recombinant protein production Functional genomics Recombinant GFP technology

a b s t r a c t Agrobacterium tumefaciens is widely used as a versatile tool for development of stably transformed model plants and crops. However, the development of Agrobacterium based transient plant transformation methods attracted substantial attention in recent years. Transient transformation methods offer several applications advancing stable transformations such as rapid and scalable recombinant protein production and in planta functional genomics studies. Herein, we highlight Agrobacterium and plant genetics factors affecting transfer of T-DNA from Agrobacterium into the plant cell nucleus and subsequent transient transgene expression. We also review recent methods concerning Agrobacterium mediated transient transformation of model plants and crops and outline key physical, physiological and genetic factors leading to their successful establishment. Of interest are especially Agrobacterium based reverse genetics studies in economically important crops relying on use of RNA interference (RNAi) or virus-induced gene silencing (VIGS) technology. The applications of Agrobacterium based transient plant transformation technology in biotech industry are presented in thorough detail. These involve production of recombinant proteins (plantibodies, vaccines and therapeutics) and effectoromics-assisted breeding of late blight resistance in potato. In addition, we also discuss biotechnological potential of recombinant GFP technology and present own examples of successful Agrobacterium mediated transient plant transformations. © 2015 Elsevier Inc. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agrobacterium virulence proteins and interacting plant proteins . . . . . . . . . . . . . . . 2.1. Activation of Agrobacterium virulence genes . . . . . . . . . . . . . . . . . . . . . 2.2. Adhesion of Agrobacterium to the plant cell surface . . . . . . . . . . . . . . . . . 2.3. T-strand formation and transport into host cytoplasm . . . . . . . . . . . . . . . . 2.4. T-complex formation and nuclear targeting . . . . . . . . . . . . . . . . . . . . . 2.5. Nuclear processing of T-complex . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Methods and applications of Agrobacterium mediated transient plant transformation . . . . . 4. Direct applications of Agrobacterium mediated transient plant transformation in biotech industry 4.1. Technical and pharmaceutical protein production . . . . . . . . . . . . . . . . . . 4.2. Effector genomics of P. infestans . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Recombinant GFP technology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions and future perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⁎ Corresponding author. Tel.: +420 58563 4978. E-mail addresses: [email protected] (P. Krenek), [email protected] (O. Samajova), [email protected] (I. Luptovciak), [email protected] (A. Doskocilova), [email protected] (G. Komis), [email protected] (J. Samaj). URL:E-mail addresses:E-mail address: http://www.cr-hana.eu/cellbiol (J. Samaj).

http://dx.doi.org/10.1016/j.biotechadv.2015.03.012 0734-9750/© 2015 Elsevier Inc. All rights reserved.

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1. Introduction The phytopathogenic gram negative bacterial species Agrobacterium tumefaciens is a causal agent of crown-gall disease in plants, which is accompanied by tumor formation on plant roots. Agrobacterium

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employs a unique virulence strategy to induce tumors; it delivers the virulent DNA molecule (Transferred DNA or T-DNA) into plant cells where it ultimately integrates into the host genome (Chilton et al, 1977). This Agrobacterium mediated genetic transformation of plants is one of the rare examples of naturally occurring transkingdom DNA transfer (Lacroix and Citovsky, 2013). The capability of Agrobacterium to integrate its own DNA into the host genome is predominantly determined by large Ti (tumor inducing) plasmid (Gelvin, 2003). Indeed, bacterial strains that are devoid of Ti plasmid are not virulent, i.e. they do not induce tumors. Moreover, virulence can be restored upon Ti plasmid acquisition. Two distinct regions harbored by Ti plasmid designated as T-DNA region and vir region are essential for tumor induction. T-DNA region is delineated by two around 25 bp imperfect repeats, designated as left and right borders (Gelvin, 2003). These regions contain genes, which encode for proteins involved in biosynthesis of plant-type hormones and opine (Zupan et al., 2000). In transformed plants, the expression of T-DNA genes induces hormone imbalance leading to cellular hyperproliferation and opine production. Opines are the exclusive source of nitrogen and energy to Agrobacterium providing a selective advantage over competing parasites (Chumakov, 2013). The vir region of Ti plasmid is not transferred to the host cell. It contains seven loci (virA, virB, virC, virD, virE, virF and virG) encoding for most of the virulence proteins (Vir proteins) required for T-DNA transport and integration into host genome (Zupan and Zambryski, 1995). Immediately upon its discovery, the unique virulence strategy of Agrobacterium attracted attention of plant biotechnologists leading to the adaptation of Agrobacterium as an unprecedented tool for genetic transformation of plants. This adaptation involved the development of binary vector system consisting of a disarmed Ti plasmid eradicated of T-DNA region and a small-easily manageable plasmid (usually up to 20 kBa) to which T-DNA region devoid of Agrobacterium genes is allocated (Gelvin, 2003). Since the T-DNA region is determined only by delineating the left and right borders and not by any other DNA sequence, virtually any type of DNA can be placed between the borders and utilized for plant transformation. It was originally shown that upon Agrobacterium infection of plant tissues, the expression of T-DNA harbored genes occurs in bimodal fashion: transient and stable (Janssen and Gardner, 1990). Transient expression usually peaks 2–4 days post infection of plant tissues and declines thereon in both the number of expressing cells and the expression level per single transformed cell (Lacroix and Citovsky, 2013). The stable expression requires T-DNA integration into host genome and when selection is applied it is characterized by increase in the expression level 10–14 days post infection of plant tissues (Janssen and Gardner, 1990). Although direct proof is lacking, indirect evidence indicates that transient expression predominantly occurs from T-DNA copies, which are not integrated into the host genome (reviewed in Lacroix and Citovsky, 2013). As the transient expression could approach high levels in infected tissues (Janssen and Gardner, 1990), it seems that Agrobacterium initially delivers much higher numbers of T-DNA copies into plant cells than the one(s) finally integrated into host genome. Following this scenario, the decrease in the transient expression peak could be explained by inherent instability of unintegrated T-DNA copies (Lacroix and Citovsky, 2013), while subsequent increase in expression level by growing number of selected cells containing stably integrated T-DNA. By convention, plants at transient expression peaks are said to be transiently transformed while tissues/plants displaying long term expression resulting from integration of T-DNA into genome are said to be stably transformed. The stable transformation of plants mediated by Agrobacterium is inheritable in the case of germline transgene transmission, thus providing a basis for the development of fully transgenic plants, in which every single cell contains a T-DNA copy integrated into its genome. Such plants display uniform and long term expression of transgene and allow for the temporal and spatial control of the transgene expression level (e.g. Bartlett et al., 2008; L. Chen et al., 2014; Clough and Bent,

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1998; Fan et al., 2008; Fillati et al., 1987; Harwood, 2012; Hiei et al., 1994, 2014; Hoekema et al., 1989; Ishida et al., 2007; Mayavan et al., 2013; Mrízová et al., 2014). Moreover, stably transformed plants can be used over many generations, although transgenes may become partially or fully inactivated by the silencing events (Fagard and Vaucheret, 2000). Within the last three decades much effort was paid to the development of Agrobacterium based stable transformation protocols for various plant species including crops. Indeed, reliable protocols are now available for the stable transformation of model plants and many crops including cereals that were originally thought to be recalcitrant to the Agrobacterium mediated transformation (e.g. Bartlett et al., 2008; Clough and Bent, 1998; Fillati et al., 1987; Hiei et al., 1994; Hoekema et al., 1989; Mrízová et al., 2014). Although other methods are suitable for plant transformation, such as protoplast or biolistic transformations, the Agrobacterium mediated transformation is preferred since plants bearing single transgene copy can be more easily obtained (e.g. Bartlett et al., 2008; Komari et al., 2004). Accordingly, vast majority of the approved genetically engineered agricultural crops have been developed using Agrobacterium (Hemmer, 2002). The importance of the green biotech sector for the global agriculture production can be demonstrated by the recent numbers: the global value of genetically modified (GM) seeds was worth of US$15.6 billion in 2013, which represents 35% of commercial seed market worth of US$45 billion (James, 2014). GM crops are widely grown in the USA and Asia, whereas in the European Union with most stringent GMO regulations, the MON 810 maize is the only GM crop approved for the commercial cultivation (Davison, 2010; James, 2014). A recent extensive global metadata analysis showed that the use of GM crops substantially reduced use of chemical pesticides while it increased crop yields and farmer profits (Klümper and Qaim, 2014). However, controversies about the health, social and environmental impact of GM crops are major reasons for the widespread negative public attitude towards this technology (Gilbert, 2013). One of the most criticized aspects is the genetic modification of commercial crops using genes imported from other species (Cressey, 2013). Nevertheless, the next-generation GM crops, which will be prepared using site specific nucleases, namely ZFNs, TALENs and CRISPR/Cas9, may harbor only precisely targeted modification of their own genomes (e.g. reviewed in Belhaj et al., 2014; K. Chen et al., 2014; Podevin et al., 2013; Voytas and Gao, 2014). Thus, such novel “transgene free” technology could possibly reduce the negative public concern on GM crops (Cressey, 2013). Although Agrobacterium mediated development of stably transformed plants is indispensable for many aspects of modern plant science and GM crops prepared in this way are still of great promise for agriculture (Wang, 2015a,b), a substantial attention is also devoted to the Agrobacterium mediated transient plant transformation, especially in recent years (please, see Sections 3, 4 and 5 below). Agrobacterium mediated transient plant transformation methods allow for rapid and scalable recombinant protein production, rapid studies of protein subcellular localization and protein– protein interactions as well as for development of functional genomics assays. In this review, we focus on Agrobacterium and plant genetic factors affecting transfer of T-DNA from Agrobacterium into the plant cell nucleus and subsequent transient transgene expression. We also outline recent methods concerning Agrobacterium based transient transformation of model plant and crop species and stress out key factors leading to their successful establishment. In order to highlight the biotechnological potential of Agrobacterium based transient plant transformation methods, three state of the art applications are discussed in detail. These involve highly efficient production of pharmaceutical proteins, applications of recombinant GFP technology and effectoromics-assisted breeding of late blight resistance in potato. 2. Agrobacterium virulence proteins and interacting plant proteins There are several steps which lead to the transport of T-DNA molecule from Agrobacterium into the host nucleus and its integration

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into host genome (Chumakov, 2013; Gelvin, 2010a). These steps include 1) activation of the Agrobacterium virulence system, 2) adhesion of Agrobacterium to the plant cell surface, 3) T-DNA excision from Ti plasmid and further processing in Agrobacterium, 4) transport of virulence proteins and T-DNA strands into the host cytoplasm, 5) TDNA complex formation and cytoplasmic trafficking, 6) transport of TDNA complex into host nucleus, 7) transport of the T-DNA complex to chromatin, 8) stripping of the proteins from the T-DNA complex prior to T-DNA integration, 9) T-DNA integration into the host genome and 10) transgene expression. These steps occur most likely in the above order during the transformation process in vivo, although activation of the Agrobacterium virulence system, its adhesion to the cell surface and T-DNA processing within Agrobacterium probably overlap partially. It is important to note that chromatin targeting of the T-complex and T-DNA integration into the host genome are not required for Agrobacterium mediated transient transformation (Gelvin, 2010b). Rather, it appears that during transient expression many T-DNA complexes are prematurely uncoated, resulting into transcriptionally active T-DNA molecules, which are rapidly degraded before they integrate into chromatin (see below). In the following text, we preferentially report on Agrobacterium and host genetic factors directly involved in the steps required for Agrobacterium mediated transient plant transformation. Obviously, up to T-complex delivery into the nucleus, stable transformation involves the same steps as transient one. Therefore any improvements in these steps will be most likely beneficial also for the stable transformation. 2.1. Activation of Agrobacterium virulence genes The activation of Agrobacterium virulence genes is dependent on the presence of phenolic compounds such as acetosyringone or hydroxyacetosyringone that are released from wounded or otherwise perturbed plants (reviewed in Bhattacharya et al., 2010). Mechanistic link between phenolic signaling molecules and virulence gene activation in Agrobacterium is provided by the two component regulatory systems comprised of VirA and VirG proteins (Gelvin, 2003; Pitzschke and Hirt, 2010, Table 1). Binding of the phenolic compound to VirA induces its autophosphorylation at histidine residue (His474). Subsequently, the phosphate group from histidine residue of VirA is transferred to aspartic acid residue in position 52 of the N-terminal

domain of VirG. Finally, activated VirG, which acts as a transcription factor, binds to the 12 bp sequence element in the promoters of all vir operons and induces transcription of vir genes. A mutagenesis screen revealed a mutation in the virG locus that led to the increase of vir gene expression in the absence of acetosyringone and VirA (Pazour et al., 1992). This mutant encodes for a constitutively active virG protein with asparagine to aspartic acid substitution at residue 54 (virGN54D). Employing this mutation, a so called ternary plant transformation system was developed (van der Fits et al., 2000). When used in transformation, the Agrobacterium strain LBA4404 bearing the ternary plasmid with virGN54D provided massive increase in transient transformation efficiencies of Arabidopsis root explants and tobacco leaf disks. In addition, even broadening of host species amenable to transformation was observed when this strain was used for the transfection of suspension cultures of various dicotyledonous species. 2.2. Adhesion of Agrobacterium to the plant cell surface Preceding pathogenesis and biofilm formation, Agrobacterium cells can adopt polar type attachment to various biotic and abiotic surfaces (Heindl et al., 2014; Matthysse, 2014; Tomlinson and Fuqua, 2009). This type of attachment depends on bacterial synthesis of extracellular polysaccharide, termed unipolar polysaccharide (UPP), which can be visualized at the attached cell pole shortly after bacterial contact with a surface (reviewed in Matthysse, 2014, Table 1). Interestingly, although it relates to visually most prominent type of attachment, characteristic by high number of attaching bacterial cells, the production of UPP is independent of pathogenesis, i.e. does not require the presence of Tiplasmid or Vir gene induction (Tomlinson and Fuqua, 2009). Recently, it has been shown that UPP synthesis and in turn also type of the Agrobacterium attachment is dependent on the concentration of calcium ions (Matthysse, 2014). UPP is synthesized under the low concentration of calcium ions coupled with phosphorus limitation and acidic pH, and it promotes polar attachment of the Agrobacterium cells. On the other hand, moderate and high concentrations of calcium ions abrogate UPP synthesis resulting in the rather very low numbers of Agrobacterium cells attached to the plant surfaces such as BY-2 protoplasts and tomato root hairs (Aguilar et al., 2011; Matthysse, 2014). In addition, Agrobacterium cells exhibit lateral attachment under elevated concentrations of calcium ions (Aguilar et al., 2011; Matthysse, 2014).

Table 1 Selected key Agrobacterium virulence proteins and structures facilitating transient plant transformation process. Phase of the transformation process

Agrobacterium protein or structure

References

Activation of the virulence system Adhesion to the plant cell surface

VirA and VirG Unipolar polysaccharide (UPP) T-pillus? (VirB2?, VirB5?) ExoR Ti plasmid, single stranded T-DNA, VirC1, VirC2, VirD1 and VirD2 T-strand, T4SS (VirB1 to VirB11), VirC1, VirD4 and VBP1

Das et al (1986), Jin et al. (1990a, 1990b, 1990c) Tomlinson and Fuqua (2009) Backert et al. (2008) Tomlinson et al. (2010) Van Haaren et al. (1987), Durrenberger et al. (1989), Young and Nester (1988), Toro et al. (1989), Scheiffele et al. (1995), Atmakuri et al. (2007), Lu et al. (2009) Vergunst et al. (2000), Cascales and Christie (2004), Christie et al. (2005) Lacroix et al. (2005), Vergunst et al. (2005), Atmakuri et al. (2007), Guo et al. (2007b), Padavannil et al. (2014) Citovsky et al. (1989, 1997), Abu-Arish et al. (2004), Grange et al. (2008), Gelvin (2012)

T-strand formationa Transport of T-strand and Vir proteins into host cytoplasm T-complex formationb and cytoplasmic trafficking Transport of T-complex into host nucleus

Uncoating of the T-complex in plant nucleusd

T-strand, VirE2 T-complex, VirD2, VirE2c, VirE3

T-complex, VirE2, VirF, VirD5

Rossi et al. (1993), Citovsky et al. (1994), Tzfira et al. (2001), Ziemienowicz et al. (2001), Tzfira et al. (2002), Lacroix et al. (2005), Li et al. (2005), Djamei et al. (2007), Grange et al. (2008), Bhattacharjee et al. (2008) Lee et al. (2008), Sakalis et al. (2013), Li et al. (2014) and Shi et al. (2014) Tzfira et al. (2004), Magori and Citovsky (2011, 2012), Zaltsman et al. (2010b, 2013), Wang et al. (2014)

? Indicate hypothetical function in the adhesion to the plant cell surface. a VirD2 covalently linked with 5′ end of the single strand (ss) T-DNA. b Existence of T-complex (T-DNA covalently linked with VirD2 and coated with VirE2) still remains hypothetical, since it has not been identified or characterized after standard infection (Gelvin, 2012). c The role of VirE2 in nuclear targeting of T-complex is rather controversial. Please see the text. d Further studies are required to clarify the process of T-complex uncoating. Please see the text.

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Intriguingly, this UPP-independent type of attachment appears to be dependent on the presence of Ti-plasmid, however, its role in bacterial attachment remains unclear (Aguilar et al., 2011; Matthysse, 2014). Hypothetically, the T-pilus, which is a filament of the VirB2 T-pillin protein with the VirB5 protein (a putative adhesin) localized at its tip, may mediate host cell contact in this case (Aly and Baron, 2007; Backert et al., 2008; Christie et al., 2005). 2.3. T-strand formation and transport into host cytoplasm Initiation of T-DNA processing involves assembly of the relaxosome protein complex at T-DNA border repeats of the Ti plasmid. In total, four vir proteins; VirC1, a member of the ParA/MinD-like ATPase protein superfamily, VirC2, VirD1 and VirD2 relaxase comprise underlying relaxosome stumbling blocks (Atmakuri et al., 2007; Lu et al., 2009; Scheiffele et al., 1995, Table 1). It was reported that VirC1 and VirC2 bind independently of VirD2 relaxase to the Ti plasmid and that binding of VirC1 is highly stimulated by the presence of VirC2 (Atmakuri et al., 2007). The binding of VirC1 and VirC2 to the Ti plasmid occurs outside the T-DNA region at conserved DNA sequence elements near the right border. In particular, VirC1 binds to a virulence enhancer designated overdrive, while VirC2 preferentially binds to a DNA element ODNC20 (overdrive without core) that adjoins overdrive from the left site (Lu et al., 2009; Toro et al., 1989; van Haaren et al., 1987). Interestingly, binding of VirD2 and VirD1 to the Ti plasmid is of transient nature and/or occurs at very low concentrations of these proteins (Atmakuri et al., 2007). Furthermore, VirC1 and VirC2 form a complex independently of other Vir proteins and at least VirC1 interacts in a pairwise fashion with VirD1 and VirD2. Moreover, VirC1 and VirC2 together highly enhance production of T-DNA transfer intermediates (see below and Atmakuri et al., 2007). Overall, all these data strongly suggest that VirC1 and VirC2 initially form a complex near the right T-DNA border and further promote border nicking via pairwise interactions with VirD2 and VirD1. Since VirD2 endonuclease cleaves both, the left and right TDNA borders (reviewed in Gelvin, 2003), the nucleosome complex presumably assembles also at the left T-DNA border of the Ti plasmid. Nevertheless, no specific protein binding elements of Ti plasmid have been described near the left T-DNA border until now. Therefore, it is likely that at least VirC1 binds to the yet undiscovered DNA element in addition to overdrive (Atmakuri et al., 2007). Moreover, the nucleosome complex at the left T-DNA border might be also partially formed by nonspecific double strand DNA binding affinity of VirC2 (Lu et al., 2009). As already mentioned, relaxosome assembly promotes nicking of the right and left T-DNA borders of the Ti plasmid by VirD2 endonuclease. It is the “lower” T-DNA strand, as designated by convention, which is nicked by VirD2. During cleavage, VirD2 covalently binds to the 5′ end of the nascent single strand (ss) T-DNA, which is subsequently released from Ti plasmid as VirD2-T-DNA nucleoprotein particle (T-strand) (reviewed in Gelvin, 2012). The translocation of the T-strand into the host cell is further guided by VirD2, which serves as a pilot protein. Several lines of evidence strongly suggest that Agrobacterium utilizes the type IV secretion system (T4SS) to translocate both T-strands and virulence proteins such as VirE2, VirE3, VirF and VirD5 into host cell (reviewed in Christie et al., 2005; Vergunst et al., 2005, Table 1). Agrobacterium T4SS, also termed VirB/VirD4 T4SS, is comprised of VirD4-coupling protein (CP) and 11 VirB proteins (VirB1 to VirB11). Whereas VirD4 CP (substrate receptor) is an inner membrane bound protein harboring the large cytoplasmic domain, 11 VirB proteins form a pore structure in the cell envelope as well as the extracellular protrusion termed T-pilus. In the presence of substrate, VirD4 CP mediates substrate docking with the VirB channel. It is indispensable for both Tstrand and VirE2 translocation into the host cell (Kumar and Das, 2002). Obviously, VirD2 should interact with the recruiting molecular machine/es to be able to guide the T-strand into host cytoplasm. Indeed, two virulence proteins were described that interact with VirD2 and mediate T-strand translocation via VirD4/VirB T4SS. At first, Atmakuri

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et al (2007) showed that VirC1 recruits the T-strands to the cell pole via interaction with VirD2. This happens independently of VirC2 and also all Ti plasmid encoded Vir proteins. Second, the interaction of VirD2 binding protein 1 (VBP1) with VirD2 was essential for Agrobacterium virulence and recruitment of the T-strand to cell poles by VBP1 was suggested (Guo et al., 2007a; Padavannil et al., 2014). Targeting of T-strands to the cell poles by VirC2 and VBP1 is in accordance with the polar localization of VirD4 and VirB components of T4SS in Agrobacterium cells (Atmakuri et al., 2007; Judd et al., 2005; Kumar and Das, 2002). As VirD4 is indispensable for polar localization of VBP1 and both VirC1 and VBP1 interact with VirD4 (Atmakuri et al., 2007; Guo et al., 2007b), it appears that VirC1 and VBP1 recruit Tcomplex directly to VirD4/VirB T4SS. In support of this statement, VBP1 was also shown to interact with VirB4 and VirB11 proteins comprising T4SS. Interestingly, not only VirD4/VirB T4SS and its substrates were shown to display polar localization, but also all four Agrobacterium replicons including the Ti plasmid locate to the cell poles (Atmakuri et al., 2007; Kahng and Shapiro, 2003). Although determined in bacterial cells cultures in the absence of plant cells, the polar localization of replicons encoding for virulence proteins and harboring T-DNA is presumably of functional importance. Atmakuri et al (2007) proposed that localized synthesis of Vir proteins (due to the presence of Ti plasmid) might simply lead to the achievement of concentration threshold required for VirD4/VirB T4SS biogenesis at the cell poles. This hypothesis was presented as an alternative to the spatial positioning of VirD4/VirB T4SS components via active recruitment mechanism. In addition, the polar localization of Agrobacterium Ti plasmid would be beneficial for translocation of T-strands into host cells. As many as 50 T-DNA transfer intermediates per cell were detected in the cytosol of induced Agrobacterium cells, indicative of successive rounds of Tstrand generation (Atmakuri et al., 2007). Thus, the localization of Ti plasmid close to the cell pole could promote more efficient and coordinated transfer of multiple T-strands via T4SS. Nevertheless, yet undiscovered feedback mechanisms might reduce the number of Tstrands produced during contact of bacterial and plant cells (Atmakuri et al., 2007). If such a feedback mechanism is revealed, its attenuation would be of great interest with respect to the enhancement of transformation efficiency and/or broadening of host range of plants susceptible to Agrobacterium transformation. Recently, polar localization of T4SS components and their substrates in induced Agrobacterium cells was challenged (Aguilar et al., 2010, 2011; Cameron et al., 2012). Using recombinant green fluorescent protein (GFP) technology and deconvolution fluorescence microscopy, VirB8 and VirD4, two membrane proteins with cytoplasmic domains, or cytoplasmic proteins VirD2, VirE2 and VirF were localized as multiple fluorescent foci around the cell surface (Aguilar et al., 2010). Moreover, immunofluorescence deconvolution microscopy of VirB proteins underlying inner membrane, periplasm and outer membrane structures of T4SS revealed similar pattern of multiple fluorescent foci (Aguilar et al., 2011). In addition, Agrobacterium cells expressing vir inducible GFP–VirB8 were shown to attach mostly laterally to tobacco protoplast cells while the immunofluorescence deconvolution microscopy of GFP– VirB8 indicated that it was also localized as multiple fluorescent foci around the cell surface of these attached cells (Aguilar et al., 2011). A new model of Agrobacterium infection strategy was proposed based on these observations, in which lateral attachment maximizes effective contact with plant cell and thus facilitates efficient transfer of T-DNA and Vir proteins via multiple T4SS localized around the cell surface (Aguilar et al., 2011). Nevertheless, above microscopical observations (Aguilar et al., 2010, 2011; Cameron et al., 2012) were performed with bacterial cells that were induced for two days on AB agar plates in the presence of acetosyringone, while previous ones regarding polar localization of Agrobacterium replicons, VirD4/VirB T4SS and their substrates were done on bacterial cells that were induced in liquid minimal media (Atmakuri et al., 2007; Guo et al., 2007b; Judd et al.,

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2005; Kumar and Das, 2002). Therefore, it might be plausible that induction of Agrobacterium cells in different types of media (liquid versus solid, acetosyringone supplemented or not) predetermine spatial distribution of VirD4/VirB T4SS and their substrates. Moreover, the cell culture experiment of Aguilar et al. (2011) was performed in the presence of high concentration of calcium ions, which is associated with the UPP-independent attachment of the Agrobacterium cells (please, see Section 2.2 and Matthysse, 2014). Thus, the observed localization of GFP–VirB8 in the laterally attached Agrobacterium cells might have been also influenced by high concentration of calcium ions in the cocultivation medium (Matthysse, 2014). Apparently, further studies are required to precisely determine the role of spatial distribution of T4SS and its relevant substrates in relation to the UPP dependent or UPP independent bacterial attachment and virulence. These studies will require time-course observations of induced bacterial cells during their interaction with plant cells, using advanced high resolution fluorescent microscopy methods and fluorescent markers of virulence and attachment process. 2.4. T-complex formation and nuclear targeting This topic has been extensively reviewed in recent years (Chumakov, 2013; Gelvin, 2010a, 2010b, 2012; Lacroix and Citovsky, 2013; Pitzschke and Hirt, 2010; Zaltsman et al., 2010a). We therefore provide only overview of major principles and focus on the most recent progress, where applicable. It remains yet unclear how is the T-DNA transported into host cell through the host plasma membrane. A role of T-pili in this process has been suggested, although a formation of protein channels consisting of VirE2 proteins in the plasma membrane may perhaps provide more reasonable explanation (reviewed in Lacroix and Citovsky, 2013). Indeed, VirE2 was shown to interact with VAP33 (VAMP (vesicle associated membrane protein) associated protein of 33 kDa)-like SNARE (SNAP (Soluble NSF Attachment Protein) REceptor) protein, a putatively integral membrane protein (Lee et al., 2012). Recombinant VirE2 protein binds cooperatively and with high efficiency in vitro to ssDNA, thus forming minimal synthetic T-complex, which consists of ssDNA fully covered with multiple VirE2 monomers (Table 1). Based on these observations, it is believed that VirE2 also associates in vivo with Tstrand (nucleoprotein complex of VirD2 and T-DNA) to generate the so-called T-complex (nucleoprotein complex of VirD2 and T-DNA coated with VirE2). One of the presumable functions of VirE2 “coat” is to protect T-DNA from degradation by nucleases inside the host cytoplasm. It appears that Agrobacterium delivers VirE2 and T-strands separately into the host cell while T-complex subsequently assembles inside the host cell cytoplasm. Cooperative binding of VirE2 to the Tstrand may serve as molecular machinery, which pull T-DNA molecule through T4SS and/or VirE2 channels into the host cytoplasm (reviewed in Lacroix and Citovsky, 2013). The nuclear targeting of T-complexes should be facilitated by interactions of VirD2 and VirE2 proteins with host plant proteins. Indeed, several proteins have been identified that interact with VirD2 and VirE2 and presumably mediate active transport of T-complexes into host nucleus via nuclear pore complexes (Table 1). VirD2 itself harbors two independent eukaryotic nuclear localization signals (NLSs); the amino terminal part of the protein contains a monopartite NLS while the carboxyl terminal part of the protein contains a bipartite NLS. Nevertheless, only a bipartite carboxyl terminal NLS of VirD2 was indispensable for import of artificial T-complexes to the plant cell nucleus (Rossi et al., 1993; Ziemienowicz et al., 2001). Apparently, the presence of eukaryotic NLSs within protein components of T-complexes suggests that Agrobacterium hijacks the host nuclear import machinery to ensure nuclear uptake of T-complexes. Indeed, VirD2 interacted with all tested Arabidopsis alpha-importin isoforms in yeast, in vitro and also in planta (Bhattacharjee et al., 2008). Moreover, VirD2 localized exclusively to the nucleus of plant and animal cells as it

formed protein complexes with various Arabidopsis alpha-importin isoforms in nuclei of tobacco protoplasts (Bhattacharjee et al., 2008; Citovsky et al., 1994). Interestingly, out of four different Arabidopsis alpha-importin mutants, only homozygous impa4 mutant showed reduced susceptibility to Agrobacterium mediated root transformation. As impa4 mutant was much less susceptible to transient transformation (in addition to the stable transformation), these data suggested importance of IMPa-4 for early steps in the Agrobacterium mediated transformation process (Bhattacharjee et al., 2008). Thus, this finding is consistent with presumable role of importin family proteins in nuclear targeting of T-complex. In addition to Arabidopsis alpha-importin family, a tomato serine/threonine protein phosphatase type 2C (PP2C) can possibly also predetermine subcellular localization of VirD2. The study of Tao et al. (2004) indicated that PP2C can act as a negative regulator of VirD2 nuclear uptake, possibly by dephosphorylating VirD2 at serine 394. Numerous studies suggested that VirE2 can act as either direct or indirect substrate of alpha-importin pathway; however, its role in nuclear targeting of T-complexes appears to be less obvious in comparison to VirD2. Two putative bipartite NLSs were identified within the central part of VirE2 suggesting that VirE2 could act as a direct target of alpha-importin pathway. Indeed, VirE2 can directly interact with several Arabidopsis alpha-importin isoforms both in yeast and in vitro (Bhattacharjee et al., 2008). Moreover, these interactions were also confirmed in planta by using bimolecular fluorescence technique (BiFC), although only overexpression of IMPa-4 promoted nuclear localization of VirE2; otherwise interactions among VirE2 and other alpha-importin isoforms appeared exclusively in the cytoplasm (Bhattacharjee et al., 2008). In addition, the capability of IMPa-4 to efficiently bind to ssDNA–VirE2 complex in vitro (via interaction with VirE2), further supported its importance for Agrobacterium mediated transformation process (Bhattacharjee et al., 2008). The interaction between VirE2 and Arabidopsis alpha-importin molecular machinery may also occur indirectly. A putative host linker protein termed VIP1 protein (VirE2 Interacting Protein 1) was identified, which can interact with both VirE2 and IMPa-1 as well as with VirE2 bound to synthetic ssDNA (Tzfira et al., 2001). VIP1 is a member of basic leucine zipper (bZIP) protein superfamily of transcription factors (TF) and normally regulates diverse responses to biotic and abiotic stresses in Arabidopsis (Djamei et al., 2007; Pitzschke et al., 2009; Tsugama et al., 2012, 2014; Wu et al., 2010). In plants, several transcription factors are direct targets of mitogen-activated protein kinase (MAPK or MPKs) signaling pathways, which play a major role in transduction of environmental signals into plant adaptive responses. Accordingly, VIP1 was shown to act as a direct substrate for Arabidopsis MPK3 (Djamei et al., 2007). Both, flagellin treatment and Agrobacterium infection activate MPK3 in Arabidopsis, which in turn phosphorylates VIP1. Phosphomimicking version of VIP1 replacing the S79 with aspartic acid (79S-D) was shown to localize predominantly to the nucleus of epidermal leaf cells of transgenic Arabidopsis plants, while nonphosphorylatable version of VIP1 replacing the S79 with alanine (79SA) remained distributed between cytoplasm and nucleus even upon flagellin treatment. These results suggested that MPK3 dependent phosphorylation of VIP1 appears to take place in the cytoplasm and subsequently triggers translocation of VIP1 to nucleus. Such an observation is in contrast, for instance, to the interaction of Arabidopsis MPK4 with WRKY33 (another transcription factor) which occurs exclusively in the nucleus (Qiu et al., 2008). As VIP1 interacts with VirE2 it seems plausible that Agrobacterium can abuse the MPK3-VIP1 signaling pathway to translocate T-complexes into the nucleus of Arabidopsis cells. Indeed, the efficiency of Agrobacterium-mediated transformation of Arabidopsis root cell culture was enhanced when wild-type VIP1 was coexpressed with the reporter, but not in the case of coexpression of nonphosphorylatable version of VIP1 (79S-A) (Djamei et al., 2007). These observations were in accordance with previous reports indicating that transgenic tobacco bearing the silenced VIP1 homologue as well as

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the Arabidopsis vip1-1 mutant showed decreased susceptibility to Agrobacterium mediated transformation, while overexpression of wild type Arabidopsis VIP1 in tobacco enhanced transformation efficiency (Li et al., 2005; Tzfira et al., 2001, 2002). Recently, however, the importance of VIP1 for the Agrobacterium mediated plant transformation was revised (Shi et al., 2014). Neither vip1-1 Arabidopsis mutant nor Arabidopsis transgenic lines overexpressing full length VIP1, VIP1 (79S-A) or VIP1 (79S-D) showed significantly altered susceptibility to Agrobacterium mediated transformation as determined with extensive root segment infection assay. Moreover, reasonable arguments were presented to explain discrepancies with previously published data. For instance, the observed unaltered susceptibility of vip 1-1 Arabidopsis mutant to Agrobacterium infection, which conflicts with the data of Li et al. (2005), is supported by high number of plants and transformed root segments analyzed as well as 5 different Agrobacterium strains tested. Furthermore, Shi et al. (2014) also noted that Tzfira et al. (2002) tested transgenic tobacco lines overexpressing Arabidopsis VIP1 cDNA, which encode for the protein lacking first 80 amino acids including serine 79. Thus, in addition to the effect of heterologous VIP1 overexpression, the enhanced susceptibility to Agrobacterium infection observed by Tzfira et al. (2002) might have been caused by the unintended N-terminal truncation of VIP1. Indeed, N-terminal truncation of first 80 amino acids promotes nonphysiological nuclear localization of VIP1 in tobacco protoplasts and onion epidermal cells (Shi et al., 2014; Tsugama et al., 2014). The decrease of susceptibility to Agrobacterium infection in tobacco lines bearing silenced VIP1 homologue (Tzfira et al., 2001) is also controversial. In this case, Shi et al. (2014) argued that the VIP1 antisense cDNA construct to generate these lines used by Tzfira et al. (2001) was not appropriate. More specifically, the tobacco homologue of VIP1 (encoding for bZIP RSG activator protein) bears no sufficient nucleotide identity to act as target for homology-based gene silencing mediated by Arabidopsis VIP1. The phenotype observed by Tzfira et al. (2001) thus might have been caused by off-target effects of VIP1 cDNA. The study of Shi et al. (2014) also showed, contrary to the observations of Djamei et al. (2007), that all VIP1, VIP1 (79S-A) and VIP1 (79S-D) equally fractionated between nucleus and cytoplasm in the cells of agroinfiltrated Nicotiana benthamiana leaves, transgenic Arabidopsis roots and Arabidopsis or tobacco BY-2 protoplasts. In this case, the observed discrepancies might be caused by the fact that Djamei et al. (2007) expressed VIP1 variants under the control of estradiol-inducible promoter in the leaves of stably transformed Arabidopsis plants, while in the study of Shi et al. (2014) VIP1 variants were constitutively overexpressed. Shi et al. (2014) also noted that conflicting localization of VIP1 variants could be also explained by different osmotic status of the plant material used in their study and the study of Djamei et al. (2007). This idea is based on the work of Tsugama et al. (2012), who showed that hypoosmotic treatment promotes nuclear uptake of VIP1 in the Arabidopsis roots. However, both VIP1 (79S-D) and wt VIP1 showed similar subcellular localization dynamics in the roots of hypoosmotically treated transgenic Arabidopsis plants (Tsugama et al., 2014). Recent studies using fluorescent protein tagging technology or split GFP technology point to predominantly cytoplasmic localization of VirE2 in plant cells even in the presence of overexpressed VIP1 and with the exception to overexpressed IMPa-4 (Bhattacharjee et al., 2008; Grange et al., 2008; Lee et al., 2008; Li et al., 2014; Sakalis et al., 2013; Shi et al., 2014). These observations further subvert the role of VirE2 in nuclear targeting of T-complexes. Thus, although VirE2 protein localization may be also affected by developmental status of particular tissue (see Gelvin, 2010a) and hypothetically also by its physiological status (please see above for VirE2 adaptor protein VIP1 and its nuclear uptake) it currently appears that T-complex nuclear uptake is predominantly guided by VirD2 protein. VirE2 protein may provide a structural scaffold for T-complex translocation through the nuclear pore and/or participate in transport via interaction with import intermediate points inside the nuclear pore (Ziemienowicz et al., 2001).

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2.5. Nuclear processing of T-complex Upon translocation from cytoplasm to the nucleus, T-DNA needs to be uncoated from its VirD2 and VirE2 protein components in order to become accessible for DNA replication and transcription molecular machinery as well as for integration of T-DNA into host genome. Within the last decade, it has been shown that T-complex is most probably disassembled in the nucleus via activity of host ubiqutin/proteasome system (UPS) (reviewed in Magori and Citovsky, 2012, Table 1). Interestingly, Agrobacterium can directly control UPS activity in plants, as it harbors Ti plasmid encoded VirF protein, which mimics the structure and function of plant F-box protein superfamily members (Tzfira et al., 2004). F-box proteins are abundant in plants with more than 700 family members predicted in the Arabidopsis genome (Xu et al., 2009) and comprise a component of the E3 ubiquitin ligase protein complex SCF (S-PHASE KINASE-ASSOCIATED PROTEIN1 (SKP1)-CULLIN1 (CUL1)-Fbox protein) or Skp1/Cullin/F-box protein complex (Wang and Deng, 2011), which recruits and ubiquitinates protein substrates for subsequent proteasome degradation. Within SCF, conserved N-terminal Fbox motif of F-box protein interacts with Skp1, whereas the variable C-terminal part of the F-box protein encompasses diverse array of domains that recognize and recruit protein substrate (Wang and Deng, 2011). The direct participation of VirF protein in plant SCF complex was initially suggested by Tzfira et al. (2004), who showed that VirF can interact with Arabidopsis Skp1 type proteins. In the same work, VirF was also shown to interact with VIP1 protein, and to promote degradation of VIP1 as well as of VirE2; however, in later case in the presence of VIP1. Thus, a direct role of Agrobacterium VirF containing plant Skp1/Cullin/F-box ligase complex in uncoating of T-complex could have been proposed. VirF itself is a targeted of plant UPS, as a part of defense mechanism acting against Agrobacterium infection. However, as a result of fascinating pathogen–host arms' race, Agrobacterium additionally secretes VirD5, which protects VirF from UPS mediated proteolysis inside the host cell (Magori and Citovsky, 2011). Agrobacterium mutated in VirF display substantially attenuated transformation efficiency in many hosts (reviewed in Magori and Citovsky, 2012). Arabidopsis and tobacco, however, show unaltered susceptibility to transformation when infected with such Agrobacterium. This indicates that at least in some plant hosts VirF independent strategy should mediate targeting of T-DNA coat for proteasome degradation. Indeed, Arabidopsis F-box protein designated VBF (VIP1-Binding F-Box protein) was shown to target VIP1 as well as its cognate VirE2 for SCFVBF dependent proteolysis (Zaltsman et al., 2010b, 2013). As VBF expression is induced by Agrobacterium infection, these observations suggest that plant defense responses directly target T-complexes for premature decoating and subsequent degradation of exposed T-DNA by nucleases. However, Agrobacterium can possibly modulate activity of VBF to promote timely deprotection of T-DNA. This idea is inferred from the recent work of Wang et al. (2014), who showed that VirD5 protein can act as a VBF competitor, thus protecting VIP1 and VirE2 from SCFVBF dependent degradation. Both, SCFVirF and SCFVBF dependent proteolyses of VirE2, require the presence of VIP1 protein as a bridging molecule between VirE2 and VirF or VirE2 and VBF, respectively. Neither VirF nor VBF can directly interact with VirE2 (Tzfira et al., 2004; Zaltsman et al., 2010b), suggesting unique role of VIP1 in the UPS mediated degradation of T-DNA protein coat. In addition, the role of proteasome in T-complex decoating appears to be obvious as proteasome inhibitor suppressed T-DNA expression (Tzfira et al., 2004). However, according to the recent study, Arabidopsis vip1-1 mutant as well as Arabidopsis plants overexpressing various VIP1 variants do not show altered susceptibility to Agrobacterium mediated transformation (Shi et al., 2014 and please see previous section). A plausible explanation for this conflicting result would be provided by the existence of yet undiscovered protein that may interconnect VirE2 with F-box proteins VirF and VBF instead of VIP1. On the other hand,

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VIP1 still could be involved in T-DNA uncoating. This suggestion is based on the nature of VIP1 gene disruption in Arabidopsis vip1-1 mutant, which was shown to produce C-terminally truncated protein encompassing first 244 amino acids of VIP1 (Li et al., 2005). The functional analysis of this truncated VIP1 protein was performed, however, because of a misannotation, the analyzed protein encompassed only 164 amino acids of VIP1, while additionally lacking first 80 amino acids. The 164 amino acids long truncated version of VIP1 could not multimerize or interact with VIP1 protein lacking first 80 amino acids, but it could bind to VirE2 (Li et al., 2005). Thus, truncated VIP1 might still support SCFVirF, VBF dependent proteolysis of T-DNA in vip1-1 Arabidopsis mutant. Future studies should verify this possibility, i.e. for instance determine if truncated version of VIP1 can interact with VirF and/or VBF. Furthermore, the evaluation of Agrobacterium mediated transformation efficiency towards Arabidopsis lines bearing VIP1 silenced via RNAi technology and/ or homozygous Arabidopsis vip1 mutants developed using targeted genome engineering techniques would definitely clarify whether VIP1 is dispensable for T-DNA uncoating and transformation process. 3. Methods and applications of Agrobacterium mediated transient plant transformation Efficient methods for Agrobacterium based transient transformation of physiologically active transgenes into several plant host species have evolved dramatically throughout the past few years (Table 2). Agroinfiltration to the leaves, co-cultivation and agroinfection using diverse strains of Agrobacteria are routinely used for transient transformation assays (Table 2). In comparison to stable Agrobacterium-mediated transformation, transient transformation methods are simpler, faster and provide convenient means for high-throughput functional screens. Tobacco and N. benthamiana leaf agroinfiltration has been widely used for its simplicity and high transformation efficiency (Voinnet et al., 2003; Yang et al., 2000, Table 2). In our laboratory we are routinely using Agrobacterium mediated transient transformation of N. benthamiana leaves for experiments aiming to visualize subcellular compartments such as nuclei, endoplasmic reticulum (ER), endosomes and cytoskeleton which are appropriately labeled by molecular markers containing GFP, YFP (yellow fluorescent protein) or RFP (red fluorescent protein) (Fig. 1). In most cases such transiently transformed leaves can provide enough recombinant protein which can be tested e.g. by immunoblot assays (Fig. 2) and used in further biochemical studies. This method is also used for testing of new research approaches. For example, the bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 endonuclease can specifically target host DNA and is thus a new approach to induce mutations at defined loci (Mussolino and Cathomen, 2013; R. Xu et al., 2014). Using Agrobacterium-mediated transient transformation, this system for gene knockout was tested in N. benthamiana (Nekrasov et al., 2013) or model and crop plants (Belhaj et al., 2013). In general, there are several key steps in transient transformation protocols that have to be followed in order to achieve sufficient expression of the gene of interest. In this part of the review, we summarize recent progress in Agrobacteriummediated transient transformation of model species and crops (Table 2). The critical aspects of successful protocols are outlined where applicable. Roles of chemicals used for transformation, physiological and biochemical factors might be crucial for achieving desirable levels of transient gene expression (Boyko et al., 2011; Kim et al., 2009; Wroblewski et al., 2005). Wroblewski et al. (2005) studied a range of factors affecting transient expression efficiency in lettuce, tomato and Arabidopsis. The developmental stage and the age of the transformed tissues were related to the levels of transient expression. First true leaves were shown to be the best option for agroinfiltration. In general, from the ontogenetic point of view, the highest level of transient expression was observed in younger cells that had completed cell division

shortly before the transformation. Agrobacterium-mediated transient transformation may elicit necrotic responses in transformed tissues as an outcome of plant defense responses. This might be circumvented by using different Agrobacterium strains for different plant species. Accordingly, a panel of Agrobacterium strains was tested for transient tomato transformation as tomato is highly prone to necrotic responses (Wroblewski et al., 2005). Among the Agrobacterium strains analysed, tumorigenic strain 1D1249 provided highest level of transformation efficiency without inducing background necrosis. Agrobacterium based transient transformation assays are not equally successful in different plant species. For instance, lettuce and tobacco are much easier transformable than Arabidopsis (Wroblewski et al., 2005). Arabidopsis is the best studied and most exploited plant in the basic research field, whereas, methods for successful and highly efficient transient transformation are not as routine as for other plant species like tobacco or lettuce. Low expression success with great variations is connected with Arabidopsis transient transformation. From 10 different Arabidopsis accessions, Ws-0 was more amenable to agroinfiltration, whereas Col-0 ecotype showed lower transformation efficacy (Wroblewski et al., 2005). Since Col-0 is the mostly studied and characterized ecotype, a considerable effort is devoted to development of assays allowing routine Agrobacterium based transient transformation. Many protocols have been tested and they are continuously improving (Bilichak et al., 2014; Li and Nebenfuhr, 2010; Li et al., 2009; Tsuda et al., 2012; Wroblewski et al., 2005; Wu et al., 2014). In 2009, Fast Agro-mediated Seedling Transformation (FAST) have been introduced and the protocol was further improved in 2010 (Li and Nebenfuhr, 2010; Li et al., 2009). FAST method is based on agro-cocultivation of Arabidopsis young seedlings with suspension of Agrobacterium cells in the presence of the surfactant. Previously, co-cultivation of Agrobacterium cells with Arabidopsis suspension cells was described (Berger et al., 2007; Koroleva et al., 2005); however, information from dedifferentiated suspension cells is limited and not relevant in the tissue context. Li et al. (2009, 2010) provided evidence that only few transformed seedlings are enough for biochemical analysis, localization and co-localization studies and also for protein–protein interaction studies. Using this method, we managed in our research group to transform several cytoskeletal markers into Arabidopsis cotyledons (Fig. 3). To verify the suitability of the method of Li et al. (2009, 2010) for functional studies of the microtubule cytoskeleton, we checked the dynamics of selected individual cortical microtubules transiently labeled with GFP fused beta-tubulin 6 and end-binding 1a protein, in pavement epidermal cells (Fig. 3). Catastrophe and rescue frequencies corresponded to published data describing microtubule dynamics in Arabidopsis plants stably transformed with microtubular GFPbased markers (Komis et al., 2014). FAST co-cultivation method was expanded to other plant species including tobacco, tomato, rice or switchgrass (Li and Nebenfuhr, 2010; Li et al., 2009). Unfortunately, FAST method was well established for Arabidopsis cotyledons but not for Arabidopsis true leaves and roots. It is possible that higher transformation efficiency in Arabidopsis root system requires wounding or more intense loosening of the cell wall. In 2010, one protocol for high-throughput transient transformation of Arabidopsis root epidermal cells was restored (Van Loock et al., 2010). Authors showed that the Agrobacterium infiltration technique that was published two years earlier and was not recommended for root localization studies (Marion et al., 2008) was sufficient for colocalization experiments in Arabidopsis root epidermis. The intensive research on plant defense systems against invading organisms and on the respective bacterial countermeasures brought important breakthrough to transient transformation procedures. Recognition of pathogen/microbe-associated molecular patterns (PAMP/MAMP) produced by invading bacteria through plant surface receptors is initiating PAMP-triggered immunity (PTI) in the host plant cells. One well studied example of PTI involves the recognition of the bacterial flagellin elicitor by receptor kinase FLAGELLIN SENSING 2 (Day et al., 2011; Chinchilla et al., 2007). To counteract the plant defense reaction, phytopathogenic gram-negative bacteria produce effector

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Table 2 An overview of Agrobacterium tumefaciens mediated transient plant transformation methods. Method

Transformed plants

Agroinfiltration Allium cepa L. (onion)

A. tumefaciens strain a

Major improvement

References

GV3101

Optimized pre-cultivation of onion bulb at 28 °C in the dark for 48 h, injection of Agrobacteria between adaxial epidermis and mesophyll, agroinfiltration medium with BAP (6-benzylaminopurine) and Silwet L-77, concentration of Agrobacterium cells optimized to OD600 = 0.10, optimized co-cultivation at 28 °C in dark for 3 days Development of transgenic Arabidopsis plants that express AvrPto (an effector protein from Pseudomonas syringae) under the control of inducible promoter. Conditional suppression of Arabidopsis immunity by AvrPto substantially enhances transient transformation efficiency Optimized leaf sampling for transformation: fourth and fifth leaf from the center of Arabidopsis plant with rosette composed of about 30 leaves Use of 3–4 day old seedlings. Addition of a sterile polyvinyl grid on a growing medium prior to sowing helps to prevent the collapse of seedlings after vacuum infiltration. Vacuum infiltration (10 mm Hg) of seedlings, repeated twice for 1 min Not obvious Selection of Agrobacterium strain. Optimized leaf sampling for transformation: second to sixth true leaf prior to full expansion Fundamental protocol for the agroinfiltration in N. benthamiana and also other plant species

Xu et al. (2014a)

Arabidopsis thaliana (thale cress)

EHA101

Arabidopsis thaliana (thale cress)

C58C1, A281, 69-2B1, 53-2A, 85-1A, (K12)

Arabidopsis thaliana (thale cress)

GV3101::pMP90

Arabidopsis thaliana (thale cress) Lactuca sativa (cultivated lettuce), Lactuca serriola (wild lettuce)

C59C1 C58C1, (K12, 1D1108)

Arabidopsis thaliana, Lactuca sativa MOG101 (cultivated lettuce), Linum usitatissimum (flax), Nicotiana benthamiana (tobacco species), N. clevelandii (tobacco species), N. tabacum, Pisum sativum (pea) Lycopersicon esculentum (tomato) 1D1249, (1D1487, 15955)

Cocultivation

Nicotiana benthamiana (tobacco species)

C58C1

Nicotiana benthamiana (tobacco species) Nicotiana tabacum (tobacco)

C58C1, 1D1249, 15955, K12, A281 EHA105

Nicotiana benthamiana (tobacco species)

GV3101

Oryza sativa L. (rice)

EHA105, LBA4404, AGL1

Pisum sativum (pea)

GV3101

Solanum tuberosum (potato)

GV3101, (LBA4404)

Triticum spp. (wheat)

LBA4404, COR308, (GV3101)

Vitis vinifera L. (grapevine)

GV3101

Vitis vinifera L. (grapevine)

C58C1

Vitis vinifera L. (grapevine)

AGL1

Arabidopsis thaliana (thale cress)

LBA4404

Arabidopsis thaliana (thale cress)

LBA4404

Selection of Agrobacterium strains, which does not elicit necrosis on tomato leaves. Optimized leaf sampling for transformation: second and older true leaves prior to full expansion A pilot use of P19 viral suppressor of post-transcriptional gene-silencing to substantially enhance transient transgene expression. Optimized leaf sampling for transformation: fourth and older true leaves prior to full expansion Establishment of agroinfiltration as an in vivo assay for the functional analysis of plant promoters and transcription factors High transient expression levels. Hyperosmotic pre-treatment of plants significantly improved the transient expression in this system. Combined effect of multiple plantlet leaf wounding, R2 basic medium supplemented with Silwet L-77 (0,01%) and cocultivation temperature (20 °C) Vacuum (0.08 MPa, 1 min) agroinfiltration of germinating pea seedlings with 2–3 cm long roots Establishment of a double Agroinfiltration procedure allowing identification of genes involved in the potato late blight resistance pathway controlled by RB gene Small incision at the site of infiltration made by needle in order to enhance efficiency of syringe assisted infiltration (genotype dependent) Repeated vacuum infiltration (15 mm Hg) for 2–3 min until the most of the detached leaf area appeared infiltrated Small cuts of leaf intervenial area before vacuum infiltration. Vacuum applied twice (−90 kPa for 2 min each) and each time quickly released to allow agroinfiltration of plantlets. Efficient syringe-assisted agroinfiltration established in hydroponically grown plants Use of pBBR1MCS virGN54D plasmid (encode for constitutively active virG) in Agrobacterium strain to induce its hypervirulence. Elimination of liquid culture step for Agrobacterium; instead direct use of Agrobacterium colonies to transform Arabidopsis suspension cultures. Simultaneous co-cultivation of Arabidopsis suspension cells with Agrobacterium containing pBBR1MCS virGN54D

Tsuda et al. (2012)

Wroblewski et al. (2005)

Marion et al. (2008)

Van Loock et al. (2010) Wroblewski et al. (2005)

Van der Hoorn et al. (2000)

Wroblewski et al. (2005)

Voinnet et al. (2003)

Wroblewski et al. (2005) Yang et al. (2000)

Zheng et al. (2012)

Andrieu et al. (2012)

Fan et al. (2011) Bhaskar et al. (2009)

Panwar et al. (2013a)

Bertazzon et al. (2012)

Visser et al. (2012)

Urso et al. (2013) Koroleva et al. (2005)

Berger et al. (2007) (continued on next page)

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Table 2 (continued) Method

Transformed plants

A. tumefaciens strain a

Arabidopsis thaliana (thale cress), EHA105 Aralia mandshurica (aralia), Betula platyphylla (birch), Nicotiana tabacum (tobacco) Phellodendron amurense (cork), Populus simonii × Populus nigra (poplar hybride), Salix matsudana (willow) GV3101::pMP90 Arabidopsis thaliana (thale cress), Lycopersicon esculentum (tomato), Oryza sativa L. (rice), Panicum virgatum (switchgrass) Arabidopsis thaliana (thale C58C1(pTiB6S3ΔT)H cress)

Agroinfection

Panicum virgatum (switchgrass)

AGL1, (EHA105, C58, GV3101)

Brachypodium distachyon (purple false brome), Hordeum vulgare (barley), Nicotiana benthamiana (tobacco species), Triticum spp. (wheat)

EHA105

Gossypium barbadense (cotton species)

GV3101, LBA4404, EHA105

Lycopersicon esculentum (tomato), Nicotiana benthamiana (tobacco species), Nicotiana tabacum (tobacco), Physalis philadelphica (tomatillo) Lycopersicon esculentum (tomato), Nicotiana benthamiana (tobacco species) Nicotiana benthamiana (tobacco species)

GV3101, (LBA4404)

Vitis vinifera L. (grapevine)

a

Major improvement plasmid and Agrobacterium expressing p19 viral suppressor. Hyperosmotic pre-treatment of plants. Optimized cocultivation conditions: Agrobacterium density of 0.4 OD600, Acetosyringone concentration of 100 μM and time laps 3 days.

References

Zheng et al. (2012)

Optimized plant developmental stage (4 or 5 days old seedlings), surfactant use (Silwet L-77 at concentration 0.005%), Agrobacterium cell density (0.5 OD600) and cocultivation time (36 h to 6 days).

Li et al. (2009)

Use of pCH32 helper plasmid in Agrobacterium strain to increase expression of VirG and VirE2. Use of buffered co-cultivation medium at pH 5.5 with AB salts. Use of EF-Tu immune receptor mutant efr-1 for transformation Combined effect of wounding pretreatments, use of three days old seedlings and addition of L-cysteine and dithiothreitol into cocultivation medium Development of Barley Stripe Mosaic Virus (BSMV) based T-DNA vector system suitable for virus induced gene silencing (VIGS) in monocot plants. Inoculation of monocot plants using infected sap from transiently transformed N. benthamiana. Establishment of efficient and specific tobacco rattle virus (TRV) mediated VIGS in cotton. Optimized light intensity (300 µmol m-2 s-1 and more), photoperiod (16 h day/8 h dark) and seedling age (cotyledon stage to the one- to two-leaf stage). Establishment of tobacco mosaic virus (TMV) based agroinfection as preferable method for the host-induced gene silencing in Bactericera cockerelli

Wu et al. (2014)

Chen et al. (2010)

Yuan et al. (2011)

Pang et al. (2013)

Wuriyanghan and Falk (2013)

GV3101

Development of Tobacco Rattle Virus (TRV) based T-DNA vector system suitable for the silencing of plant endogenous miRNA

Sha et al. (2014)

EHA105

Development of Cabbage leaf curl virus (CaLCuV) based T-DNA vector system for in planta expression of endogenous or artificial microRNAs (miRNAs) Development of Grapevine leafroll-associated virus-2 (GLRaV-2) T-DNA vector system suitable for gene expression and VIGS. Vacuum agroinfiltration of plantlets in nucerite desiccator for 10 min followed by immediate pressure release.

Tang et al. (2010)

EHA105

Kurth et al. (2012)

A. tumefaciens strains in parenthesis exhibit lower efficiency in the transfer of T-DNA.

proteins (T3E) that cause effector-triggered susceptibility (ETS) (Day et al., 2011) interfering with the PTI. T3E are directly transported to the host cells through special protein type III secretion system (T3SS). Eventually, T3E might be recognized by plant resistance proteins triggering effector-triggered immunity (ETI) (Deslandes and Rivas, 2012). Agrobacterium-mediated transformation is restricted by the plant immune system through a range of defense pathways (Hwang et al., 2010; Lee et al., 2009; Rico et al., 2010). For instance, bacterial MAMP elongation factor Tu (EF-Tu) is recognized by extracellular EF-Tu receptors and such recognition triggers plant immune response. Zipfel et al. (2006) showed that Arabidopsis EF-Tu receptor mutant efr is more amenable to Agrobacterium-mediated transient transformation in comparison to wild-type Arabidopsis Col-0 ecotype. AvrPto is one of the T3E from Pseudomonas syringae, which is targeting pathogenrecognition receptors and thus causing ETS (Xiang et al., 2008). Transgenic Arabidopsis plants expressing AvrPto under the conditional dexamethasone-inducible promoter showed much higher transient

transformation efficiency after dexamethasone treatment (Tsuda et al., 2012). This approach was successfully used for the subcellular localization of nuclear Arabidopsis protein and for identification of protein– protein interactions using Myc co-immunoprecipitation (Tsuda et al., 2012). Recently, a new protocol appeared, combining several important factors in order to achieve high transient transformation efficiency in Arabidopsis thaliana (Wu et al., 2014). The method is called AGROBEST (Agrobacterium-mediated enhanced seedling transformation). Expression of the introduced T-DNA was optimally observed in shoots, but also adequately in the root system. Interestingly, using this method, high transformation efficiency was achieved in null efr-1 mutant (lacking EF-Tu receptor). Moreover, the AGROBEST was effective even for the transient transformation of wild-type Col-0 seedlings contrary to the previously described methods of Li et al. (2009) and Marion et al. (2008). The critical factors for efficient transient transformation method in Arabidopsis were uncovered (Wu et al., 2014). First of all, disarmed Agrobacterium strain C58C1 (pTiB6S3ΔT)H was used. Authors also tested

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Fig. 1. Transient transformation of Nicotiana benthamiana leaves with A. tumefaciens harboring fluorescent organelar markers. Overview of transiently transformed leaf epidermis with A. tumefaciens carrying constructs encoding for molecular markers of organelles such as nuclei — labeled red with HTB1-RFP (A), endoplasmic reticulum — labeled red by dsRed-HDEL (B), late endosomes (arrows) — visualized by GFP-2xFYVE (C and magnification of boxed area in D), microtubules — labeled green by GFP-MBD (E) and filamentous actin — labeled green by GFP-ABD2. Red-labeled organelles in E and F are chloroplasts showing red chlorophyll autofluorescence. Scale bars = 10 μm.

the effects of seedling age, infection time and the use of antibiotic called timentin to avoid bleached lesions in Arabidopsis tissue. Another important factor was the addition of AB salts in the MS media buffered with MES to pH 5.5 during Agrobacterium infection. Studying of gene function by rapid and scalable Agrobacterium mediated transient transformation in economically important crops is of high importance especially with respect to rapid grow of transcriptomic and genomics sequence resources, which is mostly driven by availability of novel DNA sequencing technologies (Egan et al., 2012). Such functional genomics studies, virtually impossible due to practical reasons with stable transformation technologies, provide the basis for current and future development of stably engineered crops bearing, for instance, higher levels of resistance to important pests and pathogens. Most of the current protocols concerning functional gene studies in crops are based on Agrobacterium mediated transient downregulation of gene expression via RNAi (RNA interference) or VIGS (virus induced gene silencing) technology (Table 2). The major advantage of these methods is the amplification of silencing signal via siRNAs (small interfering RNAs) or recombinant viral particles, respectively, which spread from the agroinfected cells through the plant tissues and thus significantly enhance transformation efficiency. For biosafety reasons, RNAi based technologies might get a preference, however, VIGS is usually much more efficient than RNAi based gene silencing.

RNAi based gene silencing is simple and elegant method how to validate gene function through its downregulation (Helm et al., 2011). Recently, leaf vacuum agroinfiltration emerged as an efficient method for in planta RNAi based gene silencing in grapevine (Vitis vinifera L.), one of the most important crops in the Mediterranean areas (Bertazzon et al., 2012). Using this method, polygalacturonase-inhibiting protein (PGIP) that specifically inhibits fungal polygalacturonases and controls resistance to Botrytis cinerea, was efficiently and selectively transiently silenced in the leaves of in vitro grapevine plants (Bertazzon et al., 2012). Yet another assay was established in grapevine, which is based on syringe-assisted agroinfiltration of RNAi constructs into leaves (Urso et al., 2013). To verify the efficiency of the assay, a marker gene PDS (phytoene desaturase) was silenced in mildew (Uncinula necator) resistant and susceptible genotypes, respectively, and the extent of the silencing effect was evaluated by confocal microscopy and qRT-PCR. As the gene silencing efficiency was high in both genotypes tested and agroinfiltration procedure did not interfere with the U. necator infection, authors concluded that the assay might be useful for functional studies of grapevine genes involved in U. necator resistance. Finally, a functional Agrobacterium based transient expression assay was established in grapevine, which links transgene overexpression with disease resistance test (Visser et al., 2012). In particular, this assay is based on vacuum agroinfiltration of grapevine plantlets with Agrobacterium cells harboring

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Fig. 2. Transient transformation of N. benthamiana leaves with A. tumefaciens harboring 35S::GFP-AtTUB6 construct in pGWB406 (courtesy of Dr. Yoshihisha Oda, University of Tokyo, Tokyo, Japan). (A) Overview of leaf epidermis transiently expressing GFP-TUB6. (B) Maximum intensity projection of cortical microtubules visualized by GFP-TUB6. (C) Western blot analysis of GFP-TUB6 using GFP antibody. Lanes: (1) Arabidopsis thaliana plant stably transformed with GFP (35S::GFP, GFP = 27 kDa) — positive control for GFP antibody. (2) N. benthamiana transiently transformed with 35S::GFP-AtTUB6 construct (GFP-AtTUB6 = 78 kDa) in. (3) N. benthamiana — negative control for transient transformation (mock solution) and for GFP antibody. Scale bars = 20 μm.

expression vector and subsequent inoculation of agroinfiltrated plantlets with bacterial cell suspensions of Agrobacterium vitis or Xylophilus ampelinus. Using such an experimental framework, significant antimicrobial activity of D4E1 peptide against A. vitis or X. ampelinus was determined (Visser et al., 2012). Although, only the effect of gene overexpression was tested, it is tempting to speculate that such an assay would be suitable also for functional genomics studies based on transient RNAi mediated gene silencing. A highly sophisticated high throughput double agroinfiltration procedure was established in potato, which allows to determine if a particular gene is involved in the late blight resistance pathway controlled by resistance (R) gene designated RB (Bhaskar et al., 2009). In this method, Agrobacterium harboring RNAi construct is initially infiltrated into the leaves of potato constitutively overexpressing RB gene. Four days later, Agrobacterium harboring expression vector encoding for the cognate Avr factor (designated IpiO1) of RB protein (for details on R-Avr interactions please see Section 4.2) is infiltrated to the same infiltration sites. Scoring is performed 8– 10 days after first agroinfiltration. In the case of targeted gene, which is not involved in the RB pathway, a hypersensitive necrotic response occurs at the site of infiltration as a result of uninterrupted molecular events triggered by RB–IpiO interaction. However, if target gene is involved in the RB pathway, hypersensitive necrotic spot does not develop at the site of infiltration. A good system of controls is available, as SGT1, but not RAR1 is involved in the RB pathway (Bhaskar et al., 2008). Nevertheless, agroinfiltration is more challenging in cereals, which include rice and wheat that rank by production tonnage as the third and fourth most important world food crops (FAOStat, 2014, http://faostat3. fao.org/home/E). Andrieu et al. (2012) tested several conditions to establish successful agroinfiltration in rice. From different Agrobacterium strains, hyper-virulent strains EHA105, LBA4404 and AGL1 were the best choice for agroinfiltration. Preceding Agroinfiltration, leaves were

Fig. 3. Dynamics of microtubules labeled with AtTUB6-GFP (A–C) and AtEB1a-GFP (D–F). Constructs are transiently expressed in Arabidopsis thaliana 4 days old seedlings by FAST transformation method. Overviews of cotyledon epidermal cells (A, D) with selected individual microtubules (rectangles) and respective kymographs (B, E) and magnified stills at different time-points (C, F). Red arrowheads represent rescue events, white arrowheads represent catastrophe events; + and − marks point to plus and minus ends of microtubules; dotted line in kymograph shown in B highlights microtubule minus end. Catastrophes and rescues frequencies were calculated: 0.022 and 0.02 events/s, respectively for (A–C) and 0.017 and 0.015 events/s, respectively for (D–F). Scale bars for overviews (A, D) and time-laps stills (C, F) = 5 μm; scale bars for kymographs = 2 μm and 1 min (B), 2.2 min (E).

quickly wounded by a custom injection device with many microneedles (600 μm in diameter). Subsequently, wounded parts of the detached leaves were submerged into bacterial suspension. As in other assays, the usage of powerful surfactant and acetosyringone was important. Interestingly, the most critical parameter was post agroinfiltration incubation

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temperature. Optimal temperature for growth and multiplication of Agrobacteria was 25–28 °C. However, T-DNA transfer was compromised under such temperature conditions. The best results for plant cell–bacteria interaction and T-DNA transfer were obtained at 20 °C (Andrieu et al., 2012). A recent study suggested that agroinfiltration is feasible in wheat and that it can be even linked with the functional assay (Panwar et al., 2013a). Initially, Panwar et al. (2013a) optimized agroinfiltration for RNA interference in wheat using the endogenous reporter TaPDS (Triticum aestivum PHYTOENE DESATURASE) gene as a silencing target. Three different Agrobacterium strains, LBA4404, COR308 and GV3101, each harboring the same binary vector pRNAi-TaPDS, were tested for the efficiency in T-DNA delivery into leaves of wheat seedlings (cv. Thatcher). Following agroinfiltration, the highest extend of photobleaching symptoms resulting from TaPDS silencing was associated with recombinant Agrobacterium strains LBA4404 and COR308. Therefore, strain COR308 was selected for further experiments. Next, the established assay was employed for host-induced gene silencing of Puccinia triticina pathogenicity genes such as CYCLOPHILIN (PtCYC1), MAPK (PtMAPK1) and CALCINEURIN B (PtCNB). Regarding methodological aspects of this extended approach, constructs intended for the silencing of the P. triticina pathogenicity genes were initially agroinfiltrated into the leaves of wheat seedlings (cv. Thatcher). Subsequently, transformed leaves were inoculated with high dosages of P. triticina urediniospores and disease symptoms scored 10 days post inoculation. Remarkably, targeting of P. triticina pathogenicity genes was highly successful in this experiment. For the constructs tested, 51–68% reduction in pustule densities was observed, suggesting efficient migration of siRNAs from the wheat cells into fungal infection structures as well as efficient silencing of the targeted genes inside these structures. As the functional assay developed by Panwar et al. (2013a) appears to be highly efficient, it is tempting to speculate that it would be also suitable for functional genomics validations of wheat genes potentially involved in interactions with P. triticina or other wheat pathogens. It is also important to note that the agroinfiltration procedure is rather simple. Initially, small incisions are made in the leaf at the site where Agrobacterium will be infiltrated. Subsequently, a 1 ml syringe is used for gentle agroinfiltration to the leaf surface. For transient protein expression in bulb scale epidermal cells of onion the preferable method is particle bombardment (Collings, 2013). However, this method is laborintensive, costly and often associated with low transformation efficiency. Recently, a special agroinfiltration method was established achieving 43.87% transformation efficiency in living onion epidermis (K. Xu et al., 2014). Although recombinant viral replicons can be delivered into plant cells using in vitro transcription and mechanical inoculation (e.g. Panwar et al., 2013b), Agrobacterium based delivery of recombinant viral replicons is also of interest. The major advantages of such inoculation procedures include the low cost of inoculum preparation and, in the case of established agroinfection procedure, high efficiency of viral replicon delivery. The cloning capacity of plant viral vectors is usually low due to instability of larger inserts in replicating viral genomes (Yuan et al., 2011), therefore viral vectors are ideal tools especially for VIGS. In one of the recent examples, transient Agrobacterium mediated VIGS down-regulation of AGO2 and NRPD1 genes, which control DNA methylation and frequencies of DNA breaks, increased stable transformation efficiency in wild-type Arabidopsis plants (Bilichak et al., 2014). In this case, it was shown that VIGS-mediated transient downregulation of epigenetic machinery can be used as a tool for enhancement of stable floral-dip transformation (Bilichak et al., 2014). However, in addition to reverse genetics studies in model plants, Agrobacterium mediated VIGS is also the important tool for such analysis in economically important crops. Recently, the VIGS method based on agroinfection of TRV (Tobacco Rattle Virus) vector has been established in Gossypium barbadense, a cultivated cotton species that produces fiber of high quality and exhibits high level of the resistance to Verticillium dahliae (Pang et al., 2013). To establish the method, the authors tested effects of several factors on the transformation efficiency

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using the endogenous reporter PDS gene as a VIGS target. Optimization procedures showed that young seedlings (from expanding cotyledon stage up to the one- to two-leaf stage) displayed the highest silencing effects. In addition, optimal conditions for cultivation of plants after agroinfection, such as light intensity of 300 μmol m−2 s−1 and photoperiod 16 h light/8 h dark also significantly increased silencing effect. Under these optimized conditions, up to 100% of agroinfected plants displayed obvious photobleaching symptoms resulting from silencing of PDS. The high efficiency of the method was additionally verified for other three marker genes GaCLA1, GaANS and GaANR (Pang et al., 2013). Another study in Gossypium spp. successfully linked VIGS based on agroinfection of TRV vector with disease resistance test against V. dahliae (Gao and Shan, 2013). Thus, this method is suitable for high throughput reverse genetic analysis of cotton genes involved in responses to biotic stresses. Recently, also in cereals a robust Agrobacterium-based VIGS method appeared that can be linked with functional gene analysis (Yuan et al., 2011). In particular, Yuan et al (2011) adopted the tripartite Barley stripe mosaic (BSMV) vector for ligation-independent (LIC) gene cloning and for Agrobacterium-based delivery into plant cells. Since BSMV has a broad range of host species, the efficiency of the developed method could have been initially tested by agroinfection in N. benthamiana using various reporter genes as a silencing target. Indeed, the silencing efficiency in N. benthamiana was high, as inferred from the symptom development and results of the semiquantitative RT-PCR analysis. On the other hand, cereal species could not have been infected directly by Agrobacterium. BSMV harbors tripartite genome RNAα, RNAβ, and RNAγ and the method of Yuan et al (2011) relies on the simultaneous co-transformation of leaf tissues with three independent Agrobacterium clones (each harboring one of the BSMV α, β, and γ cDNAs in Ti plasmid). Thus, the failure of direct cereal agroinfection might have been caused by the demanding requirement for the successful cotransformation of cereal cells by three different Agrobacterium clones. Therefore, instead of direct agroinfection, a sap inoculation method was established, which takes advantages of high accumulation of recombinant viral particles in agroinfiltrated leaves of N. benthamiana. When infected N. benthamiana sap was used to inoculate the first true leaves of cereal species (wheat, barley and Brachypodium distachyon) the endogenous reporter gene silencing symptoms emerged on newly grown upper leaves 10–14 days post inoculum. As already mentioned, the method in discussion was also efficiently linked with functional assay. When an orthologue of Arabidopsis PMRP5 gene was downregulated in wheat using this approach and emerging upper leaves subsequently dusted with powdery mildew conidia, a significant reduction in disease symptoms was observed in infected plants (Yuan et al., 2011). 4. Direct applications of Agrobacterium mediated transient plant transformation in biotech industry Two applications, which take advantages of Agrobacteriummediated transient plant transformation for direct practical usage, are outlined in this section. These applications involve technical and pharmaceutical protein production and effector genomics of Phytophthora infestans. 4.1. Technical and pharmaceutical protein production In recent years, plants are increasingly being used as production platforms for recombinant pharmaceutical and industrial proteins because in comparison to other traditional expression platforms (e.g. microbial and mammalian ones) plants show practical, economical and safety advantages. These include reduction of cost, minimization of the risk for contamination of patients by human and animal pathogens, maximization of potential for large-scale manufacturing and flexibility (Fischer and Emans, 2000; Fischer et al., 2012, 2013;

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Gleba et al., 2007; Ma et al., 2003; Mett et al., 2008; Paul and Ma, 2011; Twyman et al., 2013). Combination of all these advantages is useful for commercial plant-based production of many recombinant proteins, including those that are not suitable for production in fermenters (Merlin et al., 2014). This newly emerging technology using plants for production of pharmaceutical recombinant proteins is named molecular farming. Moreover, plants possess the ability to perform posttranslational protein modifications which are similar to mammalian cells and include human-like complex glycosylation and disulfide bridging. Both of these modifications are often essential for biological activity of many mammalian proteins. Further, N-glycosylation pathway in plants can be targeted and manipulated. This allows specific and controlled modification and glycosylation of recombinant proteins carrying homogeneous, human-type oligosaccharides (Bosch et al., 2013). In the late 1980s, first reports on recombinant pharmaceutical proteins were published, when antibodies were produced in stably transformed tobacco (Hiatt et al., 1989). Slightly later, human serum albumin was the first recombinant pharmaceutical protein produced in transgenic tobacco and potato (Sijmons et al., 1990). From that time, plant expression platforms extended to much wider production range of biopharmaceuticals used in animal and human health care. These include cytokines, blood components, growth factors, hormones, vaccines, antibodies, industrial enzymes and dietary components (Ma et al., 2003; Yusibov et al., 2011). Production of desired recombinant proteins can occur in stably (nuclear and plastid) or transiently transformed plants (Mett et al., 2008; Tiwari et al., 2009; Xu et al., 2012). Transient transformation provides several advantages outperforming stable transformation, but the most important is a short production time. Transient expression systems offer production of target proteins in a less than two months including time required for the synthesis and expression optimization of the target gene in plants (Musiychuk et al., 2007). Transient plant transformation can be induced by recombinant plant viral vectors or bacterial binary vectors while it depends on virus expression abilities and Agrobacterium-mediated delivery. A currently more advanced expression strategy cold magnifection, utilizes advantages of Agrobacterium mediated transient plant transformation by using viral vectors for large scale production of recombinant proteins with pharmaceutical and industrial importance (Gleba et al., 2005). Magnifection is a process, which depends on Agrobacterium as a vector to deliver DNA copies encoding viral RNA/DNA replicons into diverse plant cells (Gleba et al., 2007, 2014). This method is based on vacuum infiltration for delivery of Agrobacterium carrying the expression cassette with the partially deconstructed genome of RNA virus which is directly transferred into the plant cells and cell-to-cell spread (Gleba et al., 2005, 2007, 2014; Marillonnet et al., 2005). In general, production of recombinant proteins using transient plant transformation methods was successful in N. benthamiana, Brassica rapa, Chenopodium quinoa, cowpea, alfalfa, tomato, apple and spinach (Mett et al., 2008; Tiwari et al., 2009; Xu et al., 2012). So far many pharmaceuticals including vaccines and plant-made antibodies (plantibodies) were produced using plant transient transformation methods (De Muynck et al., 2010). For example, plant production of the C5-1 (Sainsbury et al., 2009), A5 (Giritch et al., 2006) and 2G12 (Sainsbury and Lomonossoff, 2008) antibodies was achieved by using agroinfiltration. The 2G12 and αCCR5 recombinant antibodies, produced by transient expression in N. benthamiana leaves, were used as prophylactic treatment for HIV-1 (Meyers et al., 2008; Paul and Ma, 2011; Strasser et al., 2008). High-yield rapid production of hepatitis B core antigen was also performed in transiently transformed leaves of N. benthamiana. This recombinant antigen accumulated as the fulllength correctly folded product, formed disulfide-linked dimers and immunization of mice elicited production of specific antibodies (Huang et al., 2006, 2008, 2009). Further, subunit vaccine candidates against plague antigens F1 and V (Santi et al., 2006), human papilloma

virus (Massa et al., 2007), Norwalk virus (Santi et al., 2008) and smallpox (Golovkin et al., 2007) appeared to be properly folded and possessed biological activity. Recently, recombinant protein Pf38 (surface protein of the malarial parasite Plasmodium falciparum) produced in N. benthamiana leaves was used for development of vaccine against malaria (Feller et al., 2013). Immunization of mice with Pf38 resulted in generation of polyclonal antibodies with inhibitory effect on the development of the malaria parasite. Recombinant Ebola immune complex expressed in N. benthamiana leaves by using a geminiviral replicon system caused anti-Ebola virus antibody production in immunized mice (Phoolcharoen et al., 2011; Wilson et al., 2000). Plant-based oral rabies vaccine was produced in spinach using expression of the rabies virus glycoprotein and nucleoprotein in fusion to the capsid protein of alfalfa mosaic virus (Yusibov et al., 1997). Recombinant virus particles accumulated in spinach leaves and caused production of rabies virus-specific antibodies in intraperitoneally or orally treated mice (Yusibov et al., 1997) and in human volunteers after oral administration (Modelska et al., 1998; Yusibov et al., 2002). This plant-produced vaccine can be used in medical practice. Production of prophylactic and therapeutic vaccine against tuberculosis antigen ESAT6 was also achieved in transiently transformed N. benthamiana leaves (Zelada et al., 2006). Further, mice immunization with subunit vaccine against Bacillus anthracis expressed in transiently transformed N. benthamiana induced high titers of antibodies which neutralized lethal toxin (Chichester et al., 2007). Introduction of sequences encoding tumor-derived single-chain Fv epitopes of non-Hodgkin's lymphoma into N. benthamiana using tobacco mosaic virus vector resulted in production of 16 patient-specific recombinant idiotype vaccines. These are used for treatment of B-cell non-Hodgkin's lymphoma and passed the phase I clinical testing (McCormick et al., 2008). Moreover, using magnifection expression platform with a very rapid production time and high yield, 20 autologous full-idiotype IgG-based vaccines for treatment of non-Hodgkin's lymphoma patients have been produced in N. benthamiana plants. Using these vaccines protective effects against lethal tumor changes were observed in immunized mice while clinical testing with patients is currently under way (Bendandi et al., 2010). Last, but not the least, several influenza targets (H5N1 influenza HA VLP, H5N1 influenza HAI 1, H1N1 influenza HAC 1) have been expressed at high levels using plant transient expression technology (Musiychuk et al., 2007; Shoji et al., 2009, 2012; Yusibov et al., 2011). Taking together, plant transient expression systems can be used as large scale platforms for production of therapeutic proteins, antibodies and vaccines for perspective treatments of several important animal and human diseases. Especially magnifection method which is combining transient plant transformation strategy with the help of Agrobacteria and viruses provides the most advanced biotechnological potential for the future practical application in this rapidly evolving biomedical field. 4.2. Effector genomics of P. infestans Concerning global food production, potato (Solanum tuberosum ssp. Tuberosum L.) is the third most important crop following wheat and rice (Haverkort et al., 2009). Yet it encounters devastating Late Blight disease caused by oomycete hemibiothropic pathogen P. infestans. Although most “famous” as a cause of Irish famine in the middle of 19th century, Late Blight still remains a thread for contemporary potato production and one of the most important crop diseases in the world agriculture. Accordingly, one of the recent calculations estimates total loss caused by P. infestans to world potato production to be word of € 5.2 billion per annum (Haverkort et al., 2009). In developed countries, financial losses are predominantly associated with intensive plant protection (as many as 15 fungicide application per season). On the other hand, in developing and former eastern European countries, where plant protection is often inadequate or absent, Late Blight is limiting factor to potato production causing hypothetical yield losses

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word of more than € 10 billion per year (Haverkort et al., 2009). Both, high costs of fungicide plant protection (including related environmental issues) and substantial limitation of global potato production potential create long lasting demand for breeding of Late Blight resistant potato varieties. More than 180 tuber-bearing Solanum L. species from Solanaceae sect. Petota, which grow in Central and South America (Spooner and Salas, 2006), provide a wealth of so-called Rpi (Resistance to P. infestans) genes for breeding programs (e.g. Lokossou et al., 2009; Pel et al., 2009; Song et al., 2003; van der Vossen et al., 2005; van der Vossen et al., 2003; Vleeshouwers et al., 2008). However, only recently, due to advances in understanding of Oomycete–plant interactions and availability of Agrobacterium based high throughput transient transformation methods, it became possible to exploit this potential more efficiently. During early phase of infection, P. infestans, as well as other hemibiotropic and biothropic Oomycetes, secrete effector proteins inside host cells to perturb defense processes and promote host colonization. This cytoplasmic effectors involve Avr (avirulence) proteins, which are recognized by products of host Rpi genes, generally termed R proteins (Birch et al., 2006; Jones and Dangl, 2006; Marone et al., 2013). The recognition event often triggers localized defense cell death response (hypersensitive response, HR) resulting into arrest of pathogen growth (Jones and Dangl, 2006). On the other hand, in susceptible host, which doesn't harbor Rpi gene matching pathogen Avr gene/genes, infection results into host colonization and disease development. Avirulence effectors of Oomycetes harbor hypervariable C-terminal region, which determines virulence function and more conserved Nterminal region, which is essential for effector trafficking (Kamoun, 2007). This later region contains general secretory signal peptide and conserved RxLR amino acid motive that mediates effector translocation into host cell (Birch et al., 2006; Rehmany et al., 2005; Whisson et al., 2007). However, the mechanism by which Oomycete as well as fungal effectors are translocated inside host cells still remains largely unknown (Petre and Kamoun, 2014). The presence of universal host cell targeting signal (RxLR) within known Oomycete avirulence effectors (Birch et al., 2006; Rehmany et al., 2005) provided a basis for computational prediction of candidate Avr factors in different Oomycete species (Jiang et al., 2008). Many Oomycete “omics” data sets have emerged mostly in recent years such as ESTs, microarrays, RNA-seq and whole genome sequences of six species including P. infestans, and allowed identification of Avr candidate genes in various Oomycete pathogens (rewieved in Vleeshouwers and Oliver, 2014). As many as 563 RxLR genes were predicted in P. infestans genome, which is approximately 60% more than predicted in P. sojae and P. ramorum genome (Haas et al., 2009), providing a molecular rationale for its pathogenicity success. Such a high number of candidate Avr genes created demand for their functional evaluation. Indeed, high throughput functional genomics of effectors (effectoromics) emerged that uses effectors of P. infestans to probe Solanum germplasms in order to detect Rpi genes and it is also supported by functional profiling of cloned Rpi genes (Lokossou et al., 2010; Oh et al., 2009; Rietman, 2011; Rietman et al., 2012; Vleeshouwers and Oliver, 2014; Vleeshouwers et al., 2008, 2011). In the pilot study, 54 predicted RxLR effectors were probed on 10 late blight resistant Solanum genotypes leading to the identification of avirulence Avrblb1 gene along with Rpi-sto1 and Rpi-pta1, functional homologues of its cognate late blight resistance gene Rpi-blb1 (Vleeshouwers et al., 2008). It is important to note that it is an intrinsic feature of effectoromics that following gene to gene model (Jones and Dangl, 2006) novel Avr genes are identified simultaneously with their cognate Rpi genes. Another study provided functional profiling of Rpiblb2 gene (Van der Vossen et al., 2005) with 62 predicted RxLR effectors (Oh et al., 2009). This assay was based on transient reconstruction of RAvr interactions in N. benthamiana leaves and promoted rapid identification of Avrblb2. The power of effectoromics was, however, best demonstrated in the extensive study of Rietman (2011) and Rietman

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et al. (2012), who probed 17 selected resistant Solanum genotypes with 270 RxLR effector candidates. This led to the identification of 9 novel avirulence genes along with detection of 8–9 different Rpi genes in analyzed Solanum genotypes. The identity of two avirulence genes was further confirmed by reconstruction of R–Avr interactions in N. benthamiana leaves (Rietman, 2011). Technically, effectoromics relies on Agrobacterium-mediated transient expression of effector genes in small sections of leaves of Solanum germplasms, which is followed by subsequent scoring of the sites of inoculation for induced HR. Observed HR spots suggest unique R–Avr interactions, however, both false positive and false negative results can be also generated (Du and Vleeshouwers, 2014; Du et al., 2014). Therefore, positive and negative controls should be included for each genotype analyzed by this approach. Finally, reasonable response to effector should co-segregate with resistance to at least certain P. infestans isolates in the progeny of crosses between respective germplasm and susceptible parent. In particular, two different assays were established for effector profiling on Solanum sp., namely agroinfiltration and PVX agroinfection (Du and Vleeshouwers, 2014; Du et al., 2014; Rietman et al., 2012). Agroinfiltration relies on syringe based infiltration of recombinant Agrobacterium clone harboring binary effector construct into small leaf area. The method uses hypervirulent Agrobacterium strain AGL1 and ternary plasmid system encoding for constitutively active VirG gene (Du et al., 2014). The inoculum preparation involves growing of Agrobacterium in nutrient rich media and subsequent resuspension of pelleted cells in infiltration MMA medium. PVX agroinfection uses Agrobacterium strain GV3101 harboring PVX (Potato Virus X) based binary expression vector pGR106 (Torto et al., 2003; Vleeshouwers et al., 2006), which allows for extensive effector mRNA amplification and spread in leaf tissue far outside from agroinfected cells. For inoculation procedure, Agrobacteria are grown on agar plate. During inoculation, inoculum is scraped with tooth-pick tip from agar plates and subsequently delivered into the leaf by its perforation with inoculum bearing tip of tooth-pick. These simple procedures of inoculum preparation and inoculation infers high throughput format of agroinfection. Using agroinfection, at least 90 different recombinant A. tumefaciens clones can be safely screened per Solanum plant and in duplicate for the induction of localized necrosis (Vleeshouwers et al., 2006). Whereas agroinfection mainly suffers from non-specific necrotic responses and extreme resistance to PVX, agroinfiltration is more prone to generate nonspecific necrotic responses to Agrobacterium (Du and Vleeshouwers, 2014; Du et al., 2014). In addition, agroinfection is more sensitive than agroinfiltration. Thus, both methods can be used to independently confirm or complement results of each other (Du et al., 2014). Concerning effectoromics, so far we mostly pointed to its high efficiency in mining of Avr and Rpi genes (see above). However, along with this feature, effectoromics already significantly improves late blight resistance breeding in potato. All of these improvements have been excellently outlined and exemplified in recent works (Du and Vleeshouwers, 2014; Vleeshouwers and Oliver, 2014). We therefore provide only brief recapitulation here. First, effectoromics accelerates Rpi gene cloning. Using transient complementation assay in Solanum plants (Agro-co-infiltration), Rpi gene can be swiftly identified from the pool of candidates providing that cognate Avr factor is known. Second, effectors dissect functional redundancy of Rpi genes. Many wild potato species from Solanaceae sect. Petota exhibiting high levels of resistance to P. infestans are directly not crossable with potato. If an Rpi gene is originally discovered in such a species, cognate Avr factor can be used to detect its functional homologues in species amenable for direct crossing. In addition, based on their responses to effectors, Rpi genes can be precisely classified into distinct functional categories. Such a classification prevents deployment of functionally equivalent Rpi genes for breeding or cloning efforts. Third, effectors allow for accurate detection of resistance specificities in the plant material. This accounts for naturally stacked or pyramided Rpi genes, respectively,

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which are more precisely and efficiently distinguished by cognate effectors than by diagnostic sets of pathogen isolates. In addition, effectors also simplify–“mendelize” breeding for field resistance, which cannot be routinely assayed in laboratory conditions. Yet, knowledge of Avr factors and their naturally occurring allelic variants aids in engineering of cognate R genes for expanded recognition specificity. Fourth, deployment of Rpi genes in agriculture can be assisted by effectors. More precisely, spatio-temporal monitoring of Avr factors and their “virulent” allelic variants in pathogen population may aid in the decision for particular Rpi gene deployment. If done locally during season, such a monitoring strategy may also drive decisions related to plant protection, i.e. fungicides are applied only when local pathogen population is virulent towards deployed Rpi gene/genes.

5. Recombinant GFP technology Recombinant GFP technology represents preferential method for visual selection of plant transgenic cells and tissues. The main advantage of this technology is that transgenic cells, tissues and whole plants remain alive during GFP visual selection. Moreover, recombinant protein (either with or without GFP) can be targeted to diverse subcellular compartments which might help to optimize some biotech applications (e.g. production of desirable recombinant proteins in ER, their storage in the vacuole or secretion to the culture medium). The explosive development of streamlined molecular cloning procedures (e.g., Karimi et al., 2007), the identification of inherently fluorescent proteins from various marine organisms (Chudakov et al., 2010) and the vigorous development of transient and stable transformation protocols (Anami et al., 2013) led to the generation of genetically-encoded organellar tags, primarily as organellar protein fusions with the green fluorescent protein of Aequorea victoria (Prasher et al., 1992; Shimomura et al., 1962) and its spectral variants which were developed subsequently (Shaner et al., 2005). The different codon usage of transcribed gene sequences in plants required the development of adaptive strategies in order to encode functional fluorescent proteins, since the native GFP sequence contains a 84-nt stretch recognized as a cryptic intron in plants and becomes excised during mRNA processing (Haseloff et al., 1997). After developing plant codon-optimized GFP and GFP spectral variants (e.g., CFP and YFP) there has been a tremendous development of fluorescent protein applications in plant developmental and cell biology. It is finally noteworthy that advances through the development of photoswitchable and photoconvertible proteins (Wiedenmann et al., 2011; Zhou and Lin, 2013) found their way through plant cell biology increasing the potential of high precision dynamic plant cell imaging (Brown et al., 2010; Jásik and Schmelzer, 2014; Jásik et al., 2013; Lummer et al., 2011, 2013). GFP encoded under tissue or otherwise inducible specific promoters is used as marker of cell lineage fate during tissue patterning and organ development as well as to follow hormonal or stress induced gene expression (Voss et al., 2013). Even more explicitly, GFP fusions are extensively being used to follow the distribution of intracellular structures and quantitatively follow their dynamics over time. Thus GFP fusions were generated as markers of cytoskeletal organization and dynamics (Marc et al., 1998; Shaw et al., 2003; Voigt et al., 2005b), of endomembrane distribution and membrane trafficking (Geldner et al., 2009; Voigt et al., 2005a) (e.g. Fig. 1). By combining GFP with high resolution (such as total internal reflection microscopy; Vizcay-Barrena et al., 2011) or super resolution microscopies (such structured illumination microscopy; Gustafsson, 2000 or stimulated emission depletion microscopy; Hell and Wichmann, 1994), it was possible to provide subdiffraction details of microtubule dynamics, plasmodesmatal structure, cell wall biosynthesis, vesicular trafficking and diffusional or active mobility of plasma membrane proteins (e.g., Fitzgibbon et al., 2010; Kleine-Vehn et al., 2011; Komis et al., 2014; Li et al., 2013; Liesche et al., 2013).

Appropriate GFP-based biosensors have been also produced to monitor the spatiotemporal dynamics and gradients of secondary messengers such as Ca2+, reactive oxygen species, bioactive lipids or to follow changes of physiological parameters such as intracellular pH (Bencina, 2013; Choi et al., 2012; Jásik and Schmelzer, 2014; Krebs et al., 2012; Okumoto, 2012; Simon et al., 2014; Vermeer and Munnik, 2013). Protein–protein interactions that are otherwise followed genetically or biochemically (reviewed in Legrain and Rain, 2014; Rao et al., 2014) became visible and quantifiable under the microscope in living cells expressing interaction partners as FRET pairs (Förster Resonance Energy Transfer; reviewed in Muller et al., 2013) or by proximity based reconstitution of split fluorescent proteins via bimolecular fluorescence complementation (BiFC; reviewed in Kodama and Hu, 2012; Voss et al, 2013). Live cell imaging using GFP-technology is very important for elucidating various cellular processes. Most such studies rely on stable Arabidopsis transformants, the generation of which requires a substantial amount of effort and time (Clough and Bent, 1998). Transient transformations can be directly applied to Arabidopsis cell cultures by using hypervirulent Agrobacteria strains (i.e., LBA4404; Koroleva et al, 2005). Whole seedlings were successfully transiently transformed using conventional Agrobacterium strains (i.e., C58C1) by mild and short application of vacuum (Marion et al, 2008). Another typical example of utilizing transient transformation for GFP imaging involves the agroinfiltration of N. benthamiana leaves. In this case, expression of the GFP-fused molecular marker peaks at 2–4 days post-infiltration but subsequently declines to non-detectable levels via post transcriptional gene silencing (ptgs; Voinnet et al., 2003). As shown before, the problem of ptgs can be circumvented by co-expressing the p19 protein of tomato bushy stunt virus. In this case, suppression of ptgs may significantly increase the life time of transient expression. 6. Conclusions and future perspectives Undoubtedly, current Agrobacterium based transient plant transformation protocols provide unprecedented platform for diverse biotechnological applications such as recombinant protein production (plantibodies, vaccines and therapeutics) and effectoromics assisted late blight resistance breeding in potato. In addition, using recombinant GFP technology, it also allows for rapid and scalable in planta studies of protein subcellular localization and protein–protein interactions. One of the major remaining challenges is the establishment of efficient Agrobacterium based transient transformation methods in economically important cereal species, in order to allow for reliable functional genomics studies regarding involvement of candidate genes in various biotic and abiotic stresses. Recent advances, also discussed in this review, suggest that reverse genetics studies based on transient Agrobacterium mediated delivery of RNAi or virus based constructs could be feasible in cereals (Panwar et al., 2013a; Yuan et al., 2011). However, although very efficient, the BSMV based method of Yuan et al. (2011) is indirect (please see above). It remains therefore to be shown, if direct efficient Agrobacterium based VIGS method can be established in cereals. In addition, it is also of great interest, if the gene expression studies would be feasible in cereal species. Such studies will presumably rely on the use of novel Agrobacterium strains engineered towards higher efficiency of T-DNA delivery and/or next generation viral vectors. Suppression of plant immunity acting towards Agrobacterium might be also very beneficial in such analysis. Acknowledgments This work was supported by the Czech Science Foundation (GACR) grants P501/12/P455 and 14-27598P, and by grant No LO1204 (Sustainable development of research in the Centre of the Region Haná) from the National Program of Sustainability I, MEYS. PK, OŠ, AD and GK

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