Barley guanine nucleotide exchange factor HvGEF14 is an activator of the susceptibility factor HvRACB and supports host cell entry by Blumeria graminis f. sp. hordei

Abstract In barley (Hordeum vulgare), signalling rat sarcoma homolog (RHO) of plants guanosine triphosphate hydrolases (ROP GTPases) support the penetration success of Blumeria graminis f. sp. hordei but little is known about ROP activation. Guanine nucleotide exchange factors (GEFs) facilitate the exchange of ROP‐bound GDP for GTP and thereby turn ROPs into a signalling‐activated ROP‐GTP state. Plants possess a unique class of GEFs harbouring a plant‐specific ROP nucleotide exchanger domain (PRONE). Here, we performed phylogenetic analyses and annotated barley PRONE‐GEFs. The leaf epidermal‐expressed PRONE‐GEF HvGEF14 undergoes a transcriptional down‐regulation on inoculation with B. graminis f. sp. hordei and directly interacts with the ROP GTPase and susceptibility factor HvRACB in yeast and in planta. Overexpression of activated HvRACB or of HvGEF14 led to the recruitment of ROP downstream interactor HvRIC171 to the cell periphery. HvGEF14 further supported direct interaction of HvRACB with a HvRACB‐GTP‐binding CRIB (Cdc42/Rac Interactive Binding motif) domain‐containing HvRIC171 truncation. Finally, the overexpression of HvGEF14 caused enhanced susceptibility to fungal entry, while HvGEF14 RNAi provoked a trend to more penetration resistance. HvGEF14 might therefore play a role in the activation of HvRACB in barley epidermal cells during fungal penetration.


Introduction 72
In plants, cell polarity is central to cellular development and responses to external stimuli. ROPs (RHO transgenic barley plants overexpressing constitutively activated variants of either HvRACB, HvRAC1 or 98 HvRAC3 indicated a role for these ROPs in root and shoot development (Pathuri et al., 2008; GDIs prevent GDP-bound ROPs from membrane association and sequester them in the cytosol by were shown to physically interact with ROPs in a heterotetrameric complex of two ROPs with two PRONE-GEFs. After binding to ROPs, the PRONE domain facilitates a rearrangement of the GTPase 153 which leads to GDP release. Nucleotide-free ROPs and GEFs can further interact as a stable complex 154 (Berken et al., 2005;Chang et al., 2013;Gu et al., 2006;Thomas et al., 2007). The PRONE domain is 155 flanked by N-and C-terminal variable regions that likely possess regulatory functions. Especially the reactions were prepared with the Maxima 2xSYBR Green/ROX qPCR Master Mix (Thermo Scientific), 232 primers were added at 200nM concentration and 1 μl of 1/10 cDNA dilution was used. qRT-PCR was 233 run on Aria Mx3000 thermocycler (Agilent) with 40 cycles at 60°C annealing temperature for 10 234 seconds and 72°C elongation for 15 seconds and subsequent melting curve (65°C -95°C) for quality 235 control. Measurements with melting curve peaks varying from all other samples were excluded and a 236 maximum difference of 0.5 Cq between technical replicates was allowed. There were no Cq values 237 detectable for NTC of the housekeeping gene and for HvGEF14 a Cq of 36.67 was measured for NTC.

238
HvUbiquitin was measured as a housekeeping gene in all runs due to its reliable Cq values in all 239 samples that did not differ more than 2 Cq and successful usage in previous work (Schnepf et al.,    CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ;https://doi.org/10.1101https://doi.org/10. /2021 Transformed yeast was cultured in 4 ml synthetic dropout medium lacking amino acids leucine and μl 2 M LiAc and subsequently incubated for 5 min at room temperature in 100 μl 0.4 M NaOH. Pellets 260 were collected and 50 μl 4x SDS-sample buffer was added. After vortexing, samples were boiled at 95 261 °C for 5 min and shortly centrifuged before loading onto an SDS-gel (Zhang et al., 2011).

293
To test HvRACB activation status in planta, binary vectors of meGFP-HvRACB WT or meGFP-HvRACB 294 CA were co-expressed with GST-mCherry or HvCRIB46-mCherry with or without co-expression of 295 3HA-HvGEF14 via A. tumefaciens infiltration in N. benthamiana leaves.  were excited with a 561nm laser and detected at 570-610nm. GFP fluorescence was excited with a 309 488nm Argon laser and detected between 500-550nm.

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Phylogenetic analysis reveals three distinct clades of barley PRONE-GEFs 336
. CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ;https://doi.org/10.1101https://doi.org/10. /2021 To investigate guanine nucleotide exchange factors in barley, we concentrated on the well described PRONE domain-containing class of exchange factors first introduced by Berken et al. (2005). In total, 338 we identified 11 PRONE domain-encoding genes in Hordeum vulgare cv. Morex genome version 3 339 (Mascher et al., 2021). Using the MUSCLE algorithm, we aligned full-length primary amino acid sequences of these 11 barley PRONE-GEFs (Tab. 2) with 11 PRONE-GEFs of Oryza sativa, and 14 far, we first constructed a maximum-likelihood phylogenetic tree via PhyML, in which all O. sativa calculation was performed to obtain the phylogenetic tree with all three species (Fig. 1). H. vulgare reach a nomenclature that is consistent for grasses. That way, the barley PRONE-GEFs were named 348 HvGEFs 1, 2, 3a, 3b, 3c, 7, 9a, 9b, 9c, 10 and 14.

349
The resulting phylogenetic tree shows three distinct groups: clade (I), including PRONE-GEFs 1-7, 350 clade (II) clustering PRONE-GEFs 8-13 and a third clade consisting of only PRONE-GEF14 proteins from 351 the three different species (Fig. 1). Notably, in clades (I) and (II) we specifically found a clustering of The three PRONE-GEF14 sequences differ not only in their variable N-and C-terminal regions from 355 the other PRONE-GEFs but present higher levels of amino acid changes in the PRONE domain itself 356 when compared to all other PRONE-GEFs (Fig. S1, Tab. S1). PRONE-GEF14 proteins are not present in 357 the moss Physcomitrium patens or the lycophyte Selaginella moellendorffii (Eklund et al., 2010).

358
Despite its unique position in the phylogenetic tree, the overall structure of the HvGEF14 PRONE 359 domain with its three sub-domains and essential amino acids for GEF-GEF and GEF-ROP interactions 360 is conserved when compared to the other barley PRONE-GEFs (Fig. 2).

363
. CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ;https://doi.org/10.1101https://doi.org/10. /2021 addition, the variability in primary sequence of HvGEF14 is highest when compared to the consensus 368 sequence of all 11 HvPRONE-GEFs (Fig. S1, Tab. S1). However, important residues for GEF-GEF  CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint seedling shoots and epidermal peels with HvGEF14 showing the highest fragment counts in these 390 two tissues compared to all other barley PRONE-GEFs (Tab. S6).

391
The leaf epidermis of barley provides an important interface for plant-pathogen interaction. Since   CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint potentially increase GEF-binding affinity (Akamatsu et al., 2013;Berken et al., 2005;Cool et al., variants were truncated at the HvRACB CSIL motif to inhibit prenylation and membrane association in 421 yeast and hence facilitate protein accumulation in yeast nuclei. To substantiate the results, we 422 performed Western Blots, which confirmed protein stability in yeast (Fig. S5).

424
To test direct protein-protein interaction between full-length HvGEF14 and previously used variants  CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint assays in yeast and provides further evidence that HvGEF14 can also interact with the CA variant direct interaction of HvGEF14 with HvRACB-D121N in yeast (Fig. 4). The only mCherry-tagged HvRACB 448 variant, which did not provoke a reduced meGFP-HvGEF14 lifetime when compared to the control, 449 was the DN HvRACB-T20N mutant. As observed in yeast-two-hybrid assays, there was no measurable

453
To test for possible specificity of HvGEF14-ROP interaction, we also tested the interaction with

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HvRACB is partially located at the cell periphery/plasma membrane, which depends on its C-terminal 463 CAAL prenylation motif (Schultheiss et al., 2003;Schultheiss et al., 2008). HvRIC171, a barley scaffold 464 protein that has been shown to directly interact with CA but not DN barley ROP variants (Schultheiss

476
To test specific activation of the susceptibility factor HvRACB in planta, we made use of a FRET-based

HvGEF14 supports barley susceptibility towards penetration by Bgh 493
Since HvGEF14 is expressed in barley shoot epidermis and interacts with two epidermis 494 expressed HvROPs, we tested the function of the exchange factor in the interaction of barley 495 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint and Bgh. Via transient single cell overexpression or RNAi-mediated silencing of HvGEF14 in 496 barley epidermis cells and subsequent inoculation with fungal spores, we scored interaction 497 outcome for each transformed and attacked cell in at least five independent experiments. 498 On average we observed a significant increase of fungal success from 34.6% to 46.5% 499 penetration efficiency observed as haustorium formation in HvGEF14 overexpressing cells 500 when compared to an empty vector control (Fig. 8). 501 We assessed the efficacy of RNAi-mediated gene silencing to 53% according to measuring 502 the reduction of GFP fluorescence intensities of single GFP-HvGEF14 expressing barley cells 503 co-transformed with empty vector or HvGEF14 gene silencing construct, respectively (Tab. 504 S7). The knock down of HvGEF14 by RNAi led to the opposite effect of over-expression; a 505 decrease in penetration rate from 26.4% in the controls to only 16.5% in cells, in which 506 HvGEF14 was silenced by RNAi. Absolute penetration rates are a bit lower in RNAi 507 experiments due to longer incubation time between microprojectile-mediated 508 transformation and inoculation and considerably varied from experiment to experiment 509 even in the controls. Accordingly, a p-value of 0.07 was computed during statistical analysis 510 of the means (Fig. 8). However, when comparing to individual controls, all six independent 511 repetitions of the experiment showed the same trend towards reduced penetration success 512 after knock-down of HvGEF14. 513 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint

514
The barley susceptibility factor HvRACB has been studied over the last decades to 515 understand underlying molecular mechanisms of its role in support of fungal entry into 516 barley epidermal cells (Engelhardt et al., 2020). However, a critical step in this pathway may 517 be the transition from a GDP-bound, signalling inactive, towards the GTP-loaded signalling 518 activated state of HvRACB. As shown in the model species A. thaliana and in O. sativa, it is 519 apparent that plant specific PRONE-GEFs can facilitate the activation of ROP downstream 520 signalling. In this work, we therefore investigated the role of a barley PRONE-GEF candidate. 521 This revealed the epidermis-expressed and transcriptionally Bgh-regulated HvGEF14 and its 522 likely role in ROP activation and susceptibility to fungal penetration. 523 According to our phylogenetic analysis, we suggest that PRONE-GEF14 has a unique position 524 in the evolution of PRONE-GEFs. A BLAST search with the primary sequence of AtGEF14 525 reveals that the closest ortholog to PRONE-GEF14 in the moss Physcomitrium patens is 526 PpGEF1 and that there is no PRONE-GEF14 found in the moss (see Eklund et al. (2010)). In 527 addition, in the liverwort Marchantia polymorpha, only one PRONE-GEF can be found in the 528 genome. The primary sequence of this PRONE-GEF, KARAPPO, is also most similar to AtGEF1 529 Furthermore, HvRACB, HvRACD and HvROP6 had stronger transcript signals in the epidermis 537 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint compared to mesophyll in RT-PCR assays. When leaves were challenged with Bgh, several 538 susceptibility related barley ROP genes had slightly lower transcript levels compared to non-539 inoculated controls (Schultheiss et al., 2003). This gene expression profile in the epidermis is 540 reminiscent of the HvGEF14 expression in barley epidermis during Bgh attack (Fig. 3B). 541 In the model plant A. thaliana, AtGEF14 expression has also been reported in leaves via RT-542 PCR experiments. In addition, AtGEF14 expression patterns responded to a variety of abiotic This study shows direct protein-protein interaction of HvGEF14 with wild type HvRACB, CA 552 HvRACB-G15V and HvRACB-D121N (low nucleotide affinity) ( Fig. 4 and Fig. 5). In addition, we 553 report HvGEF14 interaction with wild type HvRAC1 and CA HvRAC1-G23V in yeast (Fig. S6). 554 The fact that HvGEF14 can interact with both, type I and type II ROPs suggests that it might 555 function in more than one signalling pathway. The interaction with WT ROPs provides the 556 basis for a possible role of this barley PRONE-GEF in small GTPase signalling. 557 In accordance with other studies in A. thaliana, which have previously shown interaction of 558 PRONE-GEFs with D121N-like low nucleotide affinity mutants of AtROPs (Akamatsu et al., 559 2013;Berken et al., 2005;Denninger et al., 2019;Gu et al., 2006), we could measure in 560 planta interaction of HvRACB D121N with HvGEF14. The initial discovery of the 14 A. 561 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint thaliana PRONE-GEFs was made in a yeast-two-hybrid screen with AtROP4 D121N as bait 562 (Berken et al., 2005). A similar strategy was used to find PRONE-GEFs of OsRAC1 in O. sativa 563 (Akamatsu et al., 2013). Furthermore, a global investigation into AtGEF-AtROP1 interactions 564 showed that AtGEF14 preferably interacts with a D121A/C188S mutant of AtROP1 in vitro 565 (Gu et al., 2006). This interaction of PRONE-GEFs with low nucleotide affinity ROPs might 566 stem from the fact that these ROP mutants might still cycle between activated and 567 inactivated conformations while providing stronger affinity to PRONE-GEFs (Berken et al., 568 2005). Alternatively, the D121N mutation might lead to a similar protein conformation as the 569 nucleotide free ROP, which has been crystallised in complex with the PRONE domain of 570 AtGEF8 (Thomas et al., 2007). 571 The interaction of PRONE-GEFs with other mutants of ROPs was also reported in O. sativa. 572 OsGEF1 interaction with OsRAC1 mutants was shown via split Venus fluorescence 573 complementation assays in protoplasts. Both, the constitutively activated OsRAC1-G19V as 574 well as the dominant negative OsRAC1-T24N variants were able to reconstitute fluorescence 575 of fluorescent Venus at the plasma membrane (Akamatsu et al., 2013). 576 Interaction of PRONE-GEFs with CA ROPs has also been repeatedly reported here and before 577 (e.g., Akamatsu et al. 2013;Denninger et al. 2019). Together, data support that HvGEF14 can 578 interact with HvRACB in planta. Together with data from the literature, however, a picture 579 emerges that supports that PRONE-GEFs do not only interact with ROP-GDP but possibly can 580 stay in contact to ROP-GTP after nucleotide exchange. This could have several reasons, 581 which might be further studied. One possibility is that a PRONE-GEF-ROP(GTP) complex is 582 stabilised when the ROP is arrested in a CA status. Alternatively, activated ROPs might stay in 583 contact to GEFs for a feedback regulation on ROP activity through complexes formed with 584 ROP executers as discussed before (Wu and Lew, 2013). Because PRONE-GEFs including 585 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint HvGEF14 can form dimers, GEF-GEF -ROP-GTP interaction could also recruit further ROP-586 GDP for activation (Smokvarska et al., 2021). 587 Sub-cellular localisation of signalling protein complexes is a key to ROP-mediated processes. 588 After activation, ROPs relocate to or are stabilized in their association with the plasma 589 membrane. There, they interact with downstream effectors/executers to facilitate, amongst 590 other functions, polar growth processes (Kawano et al., 2014;Poraty-Gavra et al., 2013;591 Schultheiss et al., 2003). During A. thaliana pollen tube growth, for example, the localisation 592 of activated AtROP1 to the apical plasma membrane regulates tip growth (Gu et al., 2004). 593 This specific localisation of activated AtROP1 to the membrane leads to the accumulation of 594 downstream executers like AtRIC4 in the same compartment. In presence of dominant 595 negative AtROP1, however, the RIC protein localises to the cytoplasm (Gu et al., 2005). In 596 addition, the co-expression of a ROP-GAP, which renders AtROP1 inactive, also results in the 597 relocation of AtRIC4 to the cytoplasm. In this model system of polar growth, the correct 598 localisation of activated AtROP1 as well as AtRIC4 was observed to be crucial for 599 downstream signal transduction and fine tuning of the growth process (Hwang et al., 2005). 600 To assess HvRACB signalling activity status in planta, we made use of the previously 601 published recruitment of HvRIC171 to CA HvRACB-G15V, which otherwise largely resides at 602 the plasma membrane. In the same studies, HvRIC171 was shown to be recruited to the site 603 of fungal penetration likely via endogenously activated ROPs (Schultheiss et al., 2003;604 Schultheiss et al., 2008). Via confocal microscopy, we measured higher mCherry-HvRIC171 605 localisation at the cell periphery in the presence of transiently over-expressed HvGEF14 (Fig.  606   6). Based on previous work on A. thaliana and H. vulgare RICs, this finding suggests that the 607 over-expression of HvGEF14 leads to a higher ratio of activated endogenous ROPs, which in 608 turn recruit HvRIC171 towards the cell periphery. Another way to observe ROP activation in 609 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint planta is via CRIB-based ROP activity sensors that have previously been adapted for plants 610 (Kawano et al., 2010;Wang et al., 2018;Wong et al., 2018). This so-called Ras and interactor combinations. We observed a lower GFP-lifetime when we co-expressed 618 HvGEF14, which strongly suggests that it can activate HvRACB which leads to interaction of 619 GTP-loaded HvRACB and HvCRIB46 (Fig. 7). 620 In parallel, the transient over-expression of HvGEF14 in barley epidermal cells leads to a 621 significant increase in Bgh penetration success (Fig. 8). This phenotype is similar to enhanced 622 fungal penetration during CA HvRACB-G15V over-expression (Schultheiss et al., 2003), as 623 well as the over-expression of HvRACB-downstream executers, such as HvRIC171 624 (Schultheiss et al., 2008) and HvRIPb (McCollum et al., 2020). Transient silencing of HvGEF14, 625 on the other hand, renders barley epidermis cells by trend more resistant to Bgh penetration 626 (Fig. 8). Even though the phenotype could be observed in every repetition when comparing 627 HvGEF14 RNAi with its respective control, the extend of fungal penetration varied amongst 628 repetitions so that the significance threshold of 0.05 was not met after statistical analysis. 629 Considering however, that the inoculation with Bgh naturally decreases the transcript of 630 HvGEF14 (see Fig. 3 can confirm distinct clades of PRONE-GEFs 1-7 and 8-13 as well as the placement of PRONE-663 GEF14 separated from the other two clades ( Fig. 1 and (Kim et al., 2020)). Due to high 664       (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ;https://doi.org/10.1101https://doi.org/10. /2021 Microscopy (CALM) imaging centre of TUM for providing access to the FRET-FLIM system. We are grateful to the German Research Foundation (DFG) for funding this work via SFB924.

Author contributions
RH was responsible for conception and AT, SE and RH designed the work. Data acquisition and analysis was performed by AT as well as interpretation of the data in collaboration with SE and RH. SE assisted in data acquisition for Fig. 8. AT drafted the manuscript and prepared the final version of the work after revision by SE and RH. All authors have approved the submitted version and have agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.

Data availability statement
All data generated or analysed during this study are included in this published article [and its supplementary information files]. Additional datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.    . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint

Maximum likelihood tree was calculated based on available amino acid sequences (NCBI and barley
Morex genome version3) and annotation of barley PRONE-GEFs was performed based on this tree.   Fig. S1 (Fig. S4).
. CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint    (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ;https://doi.org/10.1101https://doi.org/10. /2021 efficiency in cells with pGY1 empty vector (control OE) or after knock-down (KD) of HvGEF14 via RNAi compared to mean penetration efficiency of pIPKTA30N empty vector (control KD).
Mean values indicated with bar. Statistical significance of differences of the mean calculated with t-test in Rstudio (version 1.2.5033) after assessing for normal distribution with Shapiro-Wilk test.
. CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ;https://doi.org/10.1101https://doi.org/10. /2021  CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ;https://doi.org/10.1101https://doi.org/10. /2021 1  1  1  1  1  1  1  1  1  1  1   36  51  120  41  44  18  19  47  65  31  70 H T P Y A SP F A T P T F C S ST P V N GN SP GR A P L P P P SK K N P P A K P E I

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. CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint  Fig. S1 (Fig. S4). (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint  . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint  . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455079 doi: bioRxiv preprint