A vector system for fast-forward in vivo studies of the ZAR1 resistosome in the model plant Nicotiana benthamiana

Nicotiana benthamiana has emerged as a complementary experimental system to Arabidopsis. It enables fast-forward in vivo analyses primarily through transient gene expression and is particularly popular in the study of plant immunity. Recently, our understanding of NLR plant immune receptors has greatly advanced following the discovery of Arabidopsis ZAR1 resistosome. Here, we describe a novel vector system of 52 plasmids that enables functional studies of the ZAR1 resistosome in N. benthamiana. We showed that ZAR1 stands out among the coiled coil class of NLRs for being highly conserved across distantly related dicot plant species and confirmed NbZAR1 as the N. benthamiana ortholog of Arabidopsis ZAR1. NbZAR1 triggers autoimmune cell death in N. benthamiana and this activity is dependent on a functional N-terminal α1 helix. C-terminally tagged NbZAR1 remains functional in N. benthamiana thus enabling cell biology and biochemical studies in this plant system. We conclude that the NbZAR1 open source plasmids form an additional experimental system to Arabidopsis for in planta resistosome studies.


INTRODUCTION
Nicotiana benthamiana has developed into a popular model system in plant biology, particularly for the study of plant immunity. The appeal of N. benthamiana as an experimental system is primarily due to agroinfiltration, a rapid transient protein expression method that enables cell biology, biochemistry, protein-protein interaction analyses and other in vivo experiments (Goodin et al., 2008). In addition, N. benthamiana is a member of the asterid group of flowering plants and as such is relatively distant from the classic model plant Arabidopsis thaliana, which belongs to the rosid group. Therefore, N. benthamiana is a complementary experimental system to Arabidopsis that also allows for a broader perspective into the evolution of molecular mechanisms in dicot plants.
Plants use nucleotide-binding leucine-rich repeat (NLR) proteins to mount innate immunity against invading pathogens. NLRs are intracellular receptors that detect host cell-translocated pathogen effectors and activate a hypersensitive cell death response that is a hallmark of plant immunity. The recent elucidation of the structures of the inactive and active complexes formed by the Arabidopsis NLR protein ZAR1 (HOPZ-ACTIVATED RESISTANCE1) with its partner receptor-like cytoplasmic kinases (RLCKs) is a major breakthrough in understanding how these immune receptors are activated and function. Activated ZAR1 forms a wheel-like pentamer, termed resistosome, that undergoes a conformational switch-the death switchto expose a funnel-shaped structure formed by the N-terminal a1 helices (Wang et al., 2019a;2019b). The ZAR1 resistosome was proposed to mediate cell death directly by translocating into the plasma membrane through the funnel-like structure and perturbing membrane integrity similar to pore-forming toxins (Wang et al., 2019b). The N-terminal a1 helix matches a sequence motif known as MADA motif that is functionally conserved across ~20% of coiled-coil (CC)-type plant NLRs (Adachi et al., 2019). This indicates that the ZAR1 death switch mechanism may be widely conserved across plant species. Mutations in surface-exposed residues within the a1 helix/MADA motif impair cell death and disease resistance mediated by ZAR1 and other MADA-CC-NLRs (Wang et al., 2019b;Adachi et al., 2019).
Here, we describe a Golden Gate compatible vector system (pZA) that enables functional studies of the ZAR1 resistosome in N. benthamiana. Golden Gate enables modular cloning through assembly of DNA fragments in one-step Type IIS restriction enzyme reactions. Consistent with previous reports (Baudin et al. 2017;, we confirmed that Arabidopsis ZAR1 (AtZAR1) does not cause hypersensitive cell death in N. benthamiana when expressed on its own and that N. benthamiana carries an ortholog of ZAR1 (NbZAR1). We developed and validated the pZA plasmid collection and share it as an open source resource that should prove useful for functional and comparative studies of NLR resistosomes in plants.
Based on our experience, ZAR1 seemed atypical among NLRs for having well-defined orthologs across unrelated dicot species. To directly evaluate ZAR1 conservation relative to other CC-NLRs, we calculated the phylogenetic (patristic) distance between each of the 47 Arabidopsis CC-NLRs and their closest gene from the other plant species based on the phylogenetic tree ( Figure 1A). Interestingly, AtZAR1 stood out among the CC-NLRs as having the shortest phylogenetic distance to its four orthologs ( Figure 1B). Similarly, in a reverse analysis where we plotted the phylogenetic distance between each of the 153 N. benthamiana CC-NLRs to their closest gene from the other species, NbZAR1 again stood out as being exceptionally conserved across all five examined species relative to other N. benthamiana CC-NLRs (Figure 1-figure supplement 2). Multiple alignments of the ZAR1 orthologs from the examined species confirmed the relatively high conservation of ZAR1 with pair-wise sequence similarities ranging from 58% to 86% across the full NLR protein ( Figure  1-figure supplement 1). Overall, these analyses confirm NbZAR1 as the N. benthamiana ortholog of AtZAR1 and reveal ZAR1 as possibly the most widely conserved CC-NLR in dicot plants.

Golden Gate compatible ZAR1 plasmids for in vivo resistosome studies
The Golden Gate cloning system enables rapid and high throughput assembly of multiple sequence modules, such as promotor, terminator or tags into a common vector (Patron et al., 2015). To facilitate in vivo resistosome studies using this cloning strategy, we first introduced synonymous mutations to BpiI restriction enzyme sites in the coding sequence of AtZAR1 and NbZAR1 to make them both Golden Gate compatible and enable their transfer into a variety of Agrobacterium tumefaciens binary vectors for in planta protein expression. We cloned the genes into the Golden Gate compatible vector pUC8-Spec (pZA1, 5, 9 and 13 in Table 1). Each coding sequence was flanked by BsaI restriction enzyme sites and overhang sequences for optimal Golden Gate reactions (Table 1-figure supplement 1).

Unlike Arabidopsis ZAR1, NbZAR1 triggers autoimmune cell death in N. benthamiana.
To validate the ZAR1 constructs we generated, we first used agroinfiltration to transiently express wild-type ZAR1 and the MHD mutants AtZAR1 D489V and NbZAR1 D481V in N. benthamiana leaves ( Figure 2A). As recently reported by Baudin et al. (2019), AtZAR1 did not trigger macroscopic cell death in N. benthamiana leaves even when the full-length protein carries the D489V mutation in the MHD motif ( Figure 2B and 2C). In contrast, NbZAR1 caused macroscopic cell death when the MHD mutant NbZAR1 D481V was expressed in N. benthamiana leaves ( Figure 2D and 2E). NRC4 was used as a control MADA-CC-NLR, because its MHD mutant NRC4 D478V causes autoactive cell death in N. benthamiana leaves (Adachi et al., 2019). Our results indicate that unlike Arabidopsis ZAR1, full-length NbZAR1 can be made autoactive in N. benthamiana through mutation of the MHD motif. These results are consistent with a previous report that AtZAR1 causes hypersensitive cell death in N. benthamiana when coexpressed with the Arabidopsis RLCK AtZED1 (Baudin et al., 2017). We conclude that unlike AtZAR1, NbZAR1 is probably compatible with endogenous RLCKs and can therefore display autoactivity when expressed on its own in N. benthamiana leaves.

NbZAR1 autoimmune cell death is dependent on its N-terminal a1 helix
Next, we tested whether NbZAR1 requires an intact N-terminal a1 helix/MADA motif for triggering autoactive cell death. We expressed the double MADA/MHD mutant NbZAR1 L17E/D481V in N. benthamiana leaves using agroinfiltration and observed that the leucine 17 to glutamic acid mutation significantly reduced the cell death activity compared to NbZAR1 D481V (Figure 2D and 2E). This result indicates that an intact N-terminal a1 helix is necessary for NbZAR1 D481V autoactivity. Given that the a1 helix forms a funnelshaped structure that is a key structural component of activated ZAR1 resistosome and its translocation into the plasma membrane (Wang et al., 2019b), we conclude that the NbZAR1 D481V experimental system recapitulates the resistosome model in N. benthamiana.

C-terminally tagged NbZAR1 is functional in N. benthamiana
N. benthamiana agroinfiltration is a powerful method for in vivo cell biology, biochemistry and protein-protein interaction analyses, but this experimental system requires that the target protein remains functional and intact after fusion to an epitope or fluorescent tag (Goodin et al., 2008;Bally et al., 2018). We therefore set out to determine the degree to which NbZAR1 can tolerate protein tags (Table 1). We focused on C-terminal tags, given that Nterminal tag fusion is known to interfere with the AtZAR1 cell death activity (Wang et al., 2019b). We tested NbZAR1 D481V constructs fused to three different tags, 4xMyc, 6xHA and eGFP by agroinfiltration. In all three cases, NbZAR1 D481V retained the capacity to trigger autoactive cell death although the response was weaker than the non-epitope-tagged NbZAR1 positive control ( Figure 3A to 3F). We also showed by western blot analyses that these C-terminally tagged NbZAR1 accumulated at detectable levels in N. benthamiana leaves and generally formed a single band indicative of minimal protein degradation ( Figure 3G to 3I). We conclude that C-terminal tagging of NbZAR1 doesn't dramatically interfere with the activity and integrity of this NLR protein.

Towards in vivo studies of the ZAR1 resistosome
We conclude that the open source pZA plasmid resource we developed can serve as a valuable experimental system for in vivo resistosome studies in N. benthamiana that anti-HA and anti-GFP immunoblots of AtZAR1, NbZAR1 and the mutant proteins, total proteins were prepared from N. benthamiana leaves at 2 days after agroinfiltration. Empty vector control is described as EV. Equal loading was checked with Reversible Protein Stain Kit (Thermo Fisher). complements the Arabidopsis system and provide a foundation for future studies. The autoimmune NbZAR1 D481V mutant can be readily expressed by agroinfilitration in N. benthamiana leaves and assayed for cell death activity. NbZAR1 D481V tolerates different Cterminal tags enabling in planta cell biology, biochemical and protein-protein interactions studies of the ZAR1 resistosome. These tags do not dramatically disturb the activity of NbZAR1 D481V and may not affect recruitment of endogenous RLCK partners that are potentially necessary for its function.
The pZA toolkit complements existing assays in Arabidopsis. Of particular interest is the visualization of activated NLR oligomers in vivo. Recently, ~900 kDa complexes associated with activated AtZAR1 and another Arabidopsis MADA-CC-NLR RPP7 were detected using Blue Native polyacrylamide gel electrophoresis and gel filtration (Li et al., 2020;Hu et al., 2020). N. benthamiana agroinfiltration offers a rapid and effective alternative expression system to the Arabidopsis protoplast and transgenic expression systems used by Li et al. (2020) and Hu et al. (2020). ZAR1 stands out among CC-NLRs for being widely conserved across dicot plants but it remains unknown how ZAR1 orthologs vary in their molecular and biochemical activities. The pZA system enables evolutionary studies of the ZAR1 resistosome. Comparative analyses between AtZAR1 and NbZAR1 would help answer questions about the evolution of the resistosome as a mechanism to trigger NLR-mediated immunity. Table   Reagent
To calculate the phylogenetic (patristic) distance, we used Python script based on DendroPy (Sukumaran and Mark, 2010). We calculated patristic distances from each CC-NLR to the other CC-NLRs on the phylogenetic tree (Figure 1-source data 3) and extracted the distance between CC-NLRs of Arabidopsis or N. benthamiana to the closest NLR from the other plant species. The script used for the patristic distance calculation is available from GitHub (https://github.com/slt666666/Phylogenetic_distance_plot).

Plasmid constructions
For Golden Gate cloning system, wild-type and MHD mutant clones of AtZAR1 and NbZAR1 were synthesized through GENEWIZ Standard Gene Synthesis with synonymous mutations to BpiI restriction enzyme sites. To generate MADA motif mutants of AtZAR1 and NbZAR1, the leucine (L) 17 in the MADA motif was substituted to glutamic acid (E) by site-directed mutagenesis using Phusion High-Fidelity DNA Polymerase (Thermo Fisher). To make AtZAR1 and NbZAR1 clones for C-terminal tagging, the stop codons were removed by PCR using Phusion High-Fidelity DNA Polymerase (Thermo Fisher). Primers used for the site-directed mutagenesis and removing stop codon were listed in Supplementary file 1. All wild-type and mutant cording sequences were cloned into pCR8/GW/D-TOPO (Invitrogen) as level 0 modules (Weber et al., 2011), namely pZA1-16 (Table 1). The level 0 plasmids were then used for Golden Gate assembly with or without C-terminal tag modules, pICSL50009 (6xHA, Addgene no. 50309), pICSL50010 (4xMyc, Addgene no. 50310) or pICSL50034 (eGFP, TSL SynBio) into the binary vector pICH86988 (Engler et al., 2014), namely pZA17-52 (Table 1).

Transient gene-expression and cell death assays
Transient expression of ZAR1 wild-type and mutants in N. benthamiana were performed by agroinfiltration according to methods described by Bos et al. (2006). Briefly, four-weeks old N. benthamiana plants were infiltrated with A. tumefaciens strains carrying the binary expression plasmids. A. tumefaciens suspensions were prepared in infiltration buffer (10 mM MES, 10 mM MgCl2, and 150 μM acetosyringone, pH5.6) and were adjusted to OD600 = 0.5. Macroscopic cell death phenotypes were scored according to the scale of Segretin et al. (2014) modified to range from 0 (no visible necrosis) to 7 (fully confluent necrosis).

Protein immunoblotting
Protein samples were prepared from six discs (  Spc r : spectinomycin-resistant, Km r : kanamycin-resistant.  The phylogenetic (patristic) distance of two CC-NLR nodes between N. benthamiana and the closest NLR from the other plant species were calculated from the NB-ARC phylogenetic tree in Figure 1A. The closest patristic distances are plotted with different colours based on plant species.
(A) AtZAR1 and NbZAR1 wild-type and the mutant genes were cloned in the pUC8-Spec vector (pZA1-16). The plasmid map was made with Benchling software. (B) ZAR1 genes were cloned with the stop codon in pZA1-8 plasmids. (C) ZAR1 genes were cloned without the stop codon for C-terminal tag fusion (pZA9-16). BsaI restriction enzyme sites for Golden Gate assembly are shown as red characters. 5' and 3' overhang sequences after BsaI digestion are described as 5'OV and 3'OV, respectively.

Supplementary files
Supplementary file 1. Primers Used for generating ZAR1 Golden Gate Level 0 constructs.