Facile Labeling of Sieve Element Phloem-Protein Bodies Using the Reciprocal Oligosaccharide Probe OGA488

Sieve elements of many angiosperms contain structural phloem proteins (P-proteins) that can interact to create large P-protein bodies. P-protein bodies can occlude sieve plates upon injury but the range of functional and physiological roles of P-proteins remains uncertain, in part because of challenges in labeling and visualization methods. Here, we show that a reciprocal oligosaccharide probe, OGA488, can be used in rapid and sensitive labeling of P-protein bodies in Arabidopsis, poplar, snap bean and cucumber in histological sections. OGA488 labeling of knockouts of the two Arabidopsis P-protein-encoding genes, AtSEOR1 and AtSEOR2, indicated that labeling is specific to AtSEOR2. That protein bodies were labeled and visible in Atseor1 knockouts indicates that heterodimerization of AtSEOR1 and AtSEOR2 may not be necessary for P-protein body formation. Double labeling with a previously characterized stain for P-proteins, sulphorhodamine 101, confirmed P-protein labeling and also higher specificity of OGA488 for P-proteins. OGA488 is thus robust and easily used to label P-proteins in histological sections of multiple angiosperm species.

INTRODUCTION
Phloem sieve elements are responsible for transport of phloem sap containing photoassimilates, nutrients, signaling molecules, and hormones by bulk flow under pressure (Lucas et al., 2013;Knoblauch et al., 2016). This pressurized conduit system is fundamental to plant function and survival, but is susceptible to sap loss upon mechanical injury (Knoblauch and van Bel, 1998). Another danger is posed by phloem-feeding insects and invasive fungal hyphae that access the nutritious phloem sap (Knoblauch and van Bel, 1998;Kehr, 2006;Will and van Bel, 2006). Plants have evolved strategies to circumvent these threats. While incompletely understood, proteinaceous bodies within sieve elements represent one such mechanism that plays roles in safeguarding the phloem.
Phloem-protein (P-protein) bodies have long been recognized as conspicuous ultrastructural features within angiosperm sieve elements (Esau and Cronshaw, 1967). These proteinaceous aggregates can take various forms depending on species and developmental stage (Cronshaw, 1981;Evert, 1990). In general, for intact sieve tubes P-protein bodies do not occlude sieve plate pores and are attached to the plasma membrane to facilitate unobstructed flow within the sieve tubes and across sieve plates (Froelich et al., 2011). Upon injury, P-protein aggregates can be observed at the sieve plates, suggesting that they play a direct role in occluding sieve plates and reducing phloem sap loss (Ernst et al., 2012). Additional potential roles attributed to P-proteins include inhibiting phloem feeding insects (Kehr, 2006;Medina-Ortega and Walker, 2013) and pathogens (Pagliari et al., 2017). However, experimental tests of these various functions proposed for P-proteins have not been conclusive (Knoblauch et al., 2014).
In Arabidopsis, Arabidopsis thaliana Sieve Element Occlusion-Related 1 (AtSEOR1) and AtSEOR2 have previously been shown to encode P-proteins that heterodimerize (Anstead et al., 2012). Live imaging of labeled AtSEOR1 in intact Arabidopsis roots revealed protein structures that varied with developmental stages, including spherical agglomerates in sieve elements shortly after elongation, and filamentous structures later in development (Froelich et al., 2011). This same study combined imaging with matched flow and velocity measurements to conclude that the labeled P-protein bodies did not impede flow in the phloem (Froelich et al., 2011). Mutations in either AtSEOR1 or AtSEOR2 resulted in the loss of immunolocalization signals using an antibody recognizing filamentous protein bodies (Anstead et al., 2012). GFP-tagged AtSEOR1 could complement Atseor1 loss of function mutants but was not able to complement the ability of Atseor2 loss of function mutants to form filaments. A similar result was observed with GFP-tagged AtSEOR2 failing to complement Atseor1 mutants. However, while filaments failed to form or label, globular bodies did label within sieve elements for both complementation tests. SEOR-like proteins have also been described in poplar, and are associated with spherical P-proteins that do not respond to phloem wounding (Mullendore et al., 2018).
Efficient methods for visualizing P-proteins have been challenging. The pressurized environment of interconnected sieve elements within sieve tubes results in the effect of injury propagating long distances, and potentially disrupting ultrastructural features found in uninjured phloem. Early observations using transmission electron microscopy were often misleading, as injury and fixation during preparation for microscopy could lead to large changes in the appearance and location of P-protein bodies, which were often observed occluding sieve plates. More advanced methods have provided visualizations that are more faithful to the true appearance in planta (Truernit, 2019). However, many of these techniques are technically challenging and may be limited to plant species that can be transformed. More general methods for staining P-proteins have been described, but typically are limited to sectioned material or require some degree of dissection to provide access of dyes to sieve elements. For example, Peters et al. (2006) reported that testing of numerous commercially available dyes failed to identify a compound that uniquely labeled the specialized P-protein bodies (forisomes) of Vicia faba. However, sulphorhodamine 101 (SR101) was successfully used to label forisomes in partially dissected V. faba leaflets, despite limited membrane permeability of the dye (Peters et al., 2006).
A reciprocal oligosaccharide probe was previously described that shows highly selective binding for chitosan, a product of chitin deacetylation (Mravec et al., 2014). The oligosaccharide probe, OGA 488 , has a structure that aligns opposing charges of carboxyl groups on the probe and chitosan, and carries an Alexa Fluor 488 fluorescent label. The probe was shown to have high binding affinity for chitosan-containing portions of fungal cell walls, as well as chitosan in insect exoskeletons (Mravec et al., 2014). Plants have receptors to detect chitin, which is a well characterized elicitor of plant immune responses (Sánchez-Vallet et al., 2015). However, plants do not produce chitin and it is not a component of plant cell walls (Lee et al., 2011).
In this report, we demonstrate OGA 488 labels P-protein bodies in the phloem of four Angiosperm species. We present a simple method for labeling of fresh tissues and show that labeling in Arabidopsis is specific to AtSEOR2. We discuss biological interpretations of the binding of the chitin-mimic probe by P-protein bodies in the phloem.

MATERIALS AND METHODS
Seeds were obtained from the Arabidopsis Biological Resource Center, United States (ABRC) for Arabidopsis thaliana T-DNA insertion lines in the Columbia background for GABI-KAT-609F04 (AtSEOR1 knockout, Atseor 1-1, AT3G01680) and SALK 148614C (AtSEOR2 knockout, Atseor 2-1 AT3G01670). Wild type A. thaliana ecotype Columbia was a gift from S. Harmer, University of California Davis. Homozygous knockouts were identified by screening of seedlings from individual selfed plants carrying the T-DNA of interest. Arabidopsis seedlings were grown at 21 • C under continuous light on vertical petri plates containing Murashige-Skoog medium (Caisson Labs, MSP09-50LT) with 1% sucrose and 0.8% agar. DNA was extracted from bulks of 10 plants per plate using DNeasy Plant Mini Kit following the manufacturer's protocol (Qiagen, Germantown MD United States). Homozygosity of plant lines was determined using PCR and line-specific primers. DNA from Atseor 1-1 plants was amplified using flanking left (5'-CTCGCAACATTTCAGTGAACC-3') and flanking right (5'-CTAGGGGTAGGTGGAAACTGC-3') primers (Anstead et al., 2012) that amplify across the T-DNA insertion site to cull lines heterozygous or lacking the T-DNA. An additional amplification with a T-DNA-specific right primer (5'-CAGAACTCGCCGTAAAGACTG-3') and the flanking left primer was used to detect the presence of the T-DNA insert. The same process was carried out for Atseor 2-1, using flanking left (5'-CTGATGATCACCATGTTGCTG-3') and flanking right (5'-TCTCCGAAACTTCCATAAACG-3' primers), and a T-DNA-specific left primer (5'-CAACCCTATCTCGGGCTATTC-3') in combination with the flanking right primer. All PCR amplifications were carried out on a MyCycler Thermal Cycler (Bio-Rad, Hercules, CA) using 92 • for 30 s, followed by 40 cycles of 92 • for 15 s, 57 • for 30 s, 72 • for 70 s, and a final elongation at 72 • for 5:00 min. PCR products were run on 1.2% (w/v) agarose gels at 70 V for 1.5 h. All amplifications were carried out a minimum of three times with identical results.
For staining and imaging, each experiment used a minimum of three homozygous T-DNA insertion mutants from the Atseor 1-1, Atseor 2-1, and wildtype (Columbia) plants that were grown in soil at 21 • C under continuous light. Similarly for each experiment, six ramets of clone Populus tremula × P. alba INRA 717-1B4 (Mader et al., 2016), and six plants each of Phaseolus vulgaris and Cucumis sativus were grown in soil at 21 • C under continuous light, supplemented with fertilizer (Miracle-Gro R All Purpose Plant Food) every 2 weeks following the manufacturer's instructions. Inflorescence stem segments of bolting Arabidopsis thaliana Col-0, Atseor 1-1, and Atseor 2-1 plants were taken by cutting 2-5 cm long sections approximately 1.5 cm above the base of the stem using a razor blade. Stem sections of P. tremula × P. alba (717) hybrid were harvested from the first fully expanded internodes with secondary growth. Young stems of P. vulgaris and C. sativus were likewise harvested. Prior to sectioning, Arabidopsis stem samples were embedded in 5% agarose gel. 50 µm thick sections of Arabidopsis, INRA 717-1B4 poplar hybrids, P. vulgaris and C. sativus were prepared using a vibrating microtome (Leica Vibratome Series 1000) and placed in 50 mM MES buffer pH 5.7.
For staining with reciprocal oligosaccharide probe OGA 488, a stock solution of 1 mg/ml OGA 488 was diluted 1:1,000 in 50 mM MES pH 5.7 as previously described (Mravec et al., 2014). Sections were placed in staining buffer to incubate for 1 h in the dark at room temperature. Samples were then transferred into 1.5 ml 50 mM MES buffer to dilute unbound stain for 0.5-1 h, and then removed using a paint brush to make wet mounts slides with 50 mM MES buffer for microscopy. The used staining solution was stored in the dark at -80 • C and could be reused at least 3-4 times without noticeable degradation of signal. For double labeling with SR101 (Sigma-Aldrich S7635), sections were first stained with OGA 488 for 1 h as above, that solution was removed, and then sections were stained with SR101 at 10 µg/ml in 50 mM MES for 1 h. Sections were then washed twice with 50 mM MES and then imaged. Confocal laser scanning microscopy images were obtained with a Zeiss LSM 710 with 488 nm excitation and a 493-552 nm emission for OGA 488 and excitation 560 and 611 nm emission for SR101. The same microscope settings were used for all images compared within individual experiments. All experiments were carried out a minimum of three times.

RESULTS
Toward the goal of establishing new probes for visualizing wood formation in trees, the reciprocal oligosaccharide probe, OGA 488 , was evaluated as a potential negative control for use with other fluorescently labeled oligosaccharide probes recognizing cell wall polysaccharides. Because OGA 488 is specific for chitosan, which is not a component of plant cell walls, our expectation was a negative result. Fresh cross sections of poplar stems were prepared and probed in 50 mM MES with 1 µg/ml OGA 488 , rinsed and imaged using confocal laser scanning microscopy (see section "Materials and Methods"). Surprisingly, distinct labeling of globular bodies within the secondary phloem was seen in OGA 488 probed sections (Figure 1A), which were not seen in control sections without OGA 488 (Figure 1B). Higher magnification images showed that OGA 488 signal was confined to a subset of individual cells within the secondary phloem with relatively small diameters, consistent with sieve elements (Figure 1C), while control sections showed no signal ( Figure  1D). Similarly, longitudinal sections of poplar stems revealed OGA 488 labeling in elongated cells within the secondary phloem, typically with one or a few rounded bodies labeled within each cell ( Figure 1E) with no background staining in sections lacking OGA 488 label ( Figure 1F). Images of longitudinal sections further informs interpretation of cross sections, where the plane of optical sectioning limits detection of OGA 488 signal above or below the imaging plane. While signals detected in cross sections appear to fill the cell lumen or labeled cells, in fact signal does not extend throughout the length of the cell, and the relative size of the labeled bodies reflect not only their size but also plane of section. Higher magnification images of longitudinal sections probed with OGA 488 further confirmed the presence of the rounded bodies, but also revealed labeling of more disbursed bodies within the cell, sometimes associated with the basal ends of elongated cells (Figure 1G), while control sections lacked signal ( Figure 1H). These results are reminiscent of the morphology of P-proteins previously presented for poplar (Mullendore et al., 2018).
We next tested the hypothesis that the large intracellular bodies labeled within the secondary phloem were P-protein bodies within sieve elements. To investigate this possibility, OGA 488 was tested for fluorescent staining in cross sections of Arabidopsis inflorescence stems from T-DNA insertion lines previously shown to disrupt the function of P-Protein encoding genes AtSOER1 (AT3G01680) and AtSEOR2 (AT3G01670), and wild type controls in the same ecotype background (Columbia).  Figure 2, Arabidopsis lines homozygous for T-DNA insertions were first identified and confirmed using PCR (see section "Materials and Methods"). Fresh inflorescence stem cross sections were prepared and stained with OGA 488 and visualized with laser confocal microscopy (see section "Materials and Methods"). As shown in Figure 3A, cross sections of wild type stems showed distinct, punctate staining of globular bodies within individual cells of the primary phloem in vascular bundles. The limited number of cells with staining was consistent with the modest amount of primary phloem and number of sieve elements in Arabidopsis inflorescence stems in comparison to the secondary phloem of poplar. No signal or background fluorescence was detected for wild type stems without OGA 488 label (Figure 3B), indicating highly specific staining. Staining of T-DNA insertion line GABI-KAT 609F04 disrupting AtSEOR1 with OGA 488 showed similar results as wildtype, with punctate staining in the primary phloem ( Figure 3C). In contrast, no OGA 488 staining was detected for T-DNA insertion line SALK 148614C disrupting AtSEOR2 (Figure 3D). We thus conclude that OGA 488 staining is the result of interactions specific to AtSEOR2, and that OGA 488 does not interact with AtSEOR1.

As shown in
OGA 488 staining was compared to a previously described dye, SR101, shown to stain P-protein containing forisomes in Vicia faba (Peters et al., 2006). Double labeling of cross sections of poplar stems with OGA 488 and SR101 is shown for increasing magnifications in Figure 4. OGA 488 staining (Figures 4A-G) showed punctate labeling of individual cells within secondary phloem. SR101 labeling (Figures 4B-H) also showed punctate signals from individual cells in secondary phloem, but additionally labeled secondary cell walls within secondary xylem, as well as cytoplasm and cell wall constituents in cells of the cortex, rays, and primary xylem. Overlay of OGA 488 and SR101 images (Figures 4C-I) showed overlapping signal for OGA 488 and SR101. Thus, SR101 labeling provided additional support for the notion that OGA 488 labels P-proteins, and also shows that OGA 488 is highly specific in labeling P-protein bodies in contrast to this alternative probe. Longitudinal sections of poplar stems similarly showed very specific labeling of P-proteins with OGA 488 overlapping with SR101, but with higher specificity. P-protein aggregates were labeled near sieve plates (Figure 4L), likely indicating injury of the labeled sieve elements during sectioning. Longitudinal sections also inform the interpretation of cross sections, where detection of P-protein aggregates is limited to the plane of optical sectioning.
To further test the ability of OGA 488 to specifically stain P-proteins in additional species, double labeling of stems of Phaseolus vulgaris and Cucumis sativus was tested using OGA 488 and SR101 (Figure 5). Similar to poplar and Arabidopsis, cross sections of both P. vulgaris ( Figure 5A) and C. sativus ( Figure 5D) had highly localized labeling of cells within the phloem but no other regions with OGA 488 , while SR101 labeled similar structures but also labeled secondary cell walls of xylem and other cytoplasmic components (Figures 5B,E). Overlay of OGA 488 and SR101 signals showed good colocalization, again suggesting that OGA 488 shows high specificity and can be used across a variety of species.

DISCUSSION
We found that the OGA 488 reciprocal oligosaccharide probe gave reproducible and highly specific staining of P-protein bodies in poplar, Arabidopsis, snap bean and cucumber stems. This was an unexpected result, as the OGA 488 probe was designed to reciprocally bind to chitin, and plant cells do not make chitin. Using previously characterized loss of function mutants for Arabidopsis P-protein components AtSEOR1 and AtSEOR2, we found that AtSEOR2 is responsible for binding OGA 488 . As discussed below, our results provide a new method for rapidly labeling P-protein bodies in sectioned material and raise questions about the significance of carbohydrate binding in the function of P-protein bodies.
Labeling of P-protein bodies using OGA 488 was robust and easy to execute in Arabidopsis, poplar, snap bean and cucumber stems. It should be noted that in the experiments here, freshly sectioned stem material was used and thus the morphology of P-proteins would be as expected for wounded phloem, and does not reflect the native morphology expected for intact, living cells. The labeling of P-protein bodies with OGA 488 colocalized with a previously described labeling method using SR101 (Peters et al., 2006). The OGA 488 labeling was highly specific for P-proteins in poplar (Figure 4) bean and cucumber (Figure 5), while SR101 also labeled additional structures non-specifically. Additionally, the probe proved to be stable in our hands, allowing the same staining solution to be reused multiple times without obvious degradation of signal or change in specificity. It should be noted that staining here was in sectioned stem tissues. The morphology of the P-protein bodies stained thus likely represent what is seen when the phloem is wounded and pressure is relieved, and thus does not reflect the native form of P-protein bodies in unwounded tissues. We did not succeed in labeling P-proteins robustly in intact (unsectioned) Arabidopsis roots using OGA 488 (data not shown), suggesting the probe is not cell permeant or at least not capable of penetrating multiple cell layers to reach the phloem. This limitation could potentially be overcome by those wanting to achieve high resolution imaging of native P-protein morphology using OGA 488 by partial dissection of tissues (e.g., Peters et al., 2006), and/or new approaches for delivery of cell-impermeable probes into live cells (e.g., Zhang et al., 2019). However, the ease of the assay described here could be extended to many applications and questions not requiring native P-protein morphology including identification of sieve elements, or how P-protein bodies change in different genotypic backgrounds, in response to insect or pathogen interactions, or assays of physical wounding or biochemical responses. This is in contrast to other technically challenging approaches (Truernit, 2019) designed to maintain P-protein integrity for immunolocalization and/or electron microscopy (Hunziker and Schulz, 2019), or transgenic expression of GFPtranslational fusions for live cell imaging (Pélissier et al., 2008;Cayla et al., 2019).
The target of labeling OGA 488 was investigated here using previously described knockout mutants for Arabidopsis P-protein-encoding AtSEOR1 and AtSEOR2 (Anstead et al., 2012). Signal for OGA 488 was present in atseor1 mutants but lacking in atseor2 mutants, allowing us to conclude that AtSEOR2 binds OGA 488 . How OGA 488 labels AtSEOR2 is an intriguing question, and while not directly addressed in the present work it is possible that binding is through the reciprocal carbohydrate bonding approach originally designed for the probe (Mravec et al., 2014), which would predict that AtSEOR2 is a glycoprotein decorated with carbohydrate capable of interacting with OGA 488 . Previous reports investigating the structure and function of P-proteins ascribed in vitro carbohydrate-binding lectin activity to Phloem Protein 2 (PP2) from Cucurbita maxima and Cucumis melo, which bound to chitin columns (Sabnis and Hart, 1978;Allen, 1979;Read and Northcote, 1983;Dinant et al., 2003). However, PP2-like proteins are phylogenetically unrelated to the AtSEOR2, and AtSEOR2 has not been reported to be glycosylated or routed through the secretory pathway. Alternatively, the AtSEOR2 protein may selectively bind the probe through protein-carbohydrate interactions. To our knowledge, carbohydrate-binding lectin properties have not been previously described for AtSEOR2 and could now be further investigated in future research.
It is intriguing to ask what the functional significance of lectinlike carbohydrate binding of AtSEOR2 to OGA 488 could be.
Previous research has suggested various functions of P-proteins, including directly interacting with or otherwise aggravating insect, fungal, or viral pests of the phloem, but irrefutable assignment of function to P-proteins remains elusive (Knoblauch et al., 2014). Lectin-like carbohydrate binding by AtSEOR2 to the OGA 488 could be fortuitous, or may reflect a normal function of the protein. Chitin present in the stylets of phloem-feeding insects and the wall of fungal pathogens both present potential binding targets for AtSEOR2.
The biological complexity and extreme economic costs of phloem-feeding insects and associated vectored pathogens (Jiang et al., 2019) requires new tools, concepts and insights. We hope that the labeling procedure reported here will be a useful addition to the tools available for the study of P-proteins, and inspire new research into the nature of carbohydrate binding by AtSERO2.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
AG conceived and managed the study. AL made the initial observation of OGA488 labeling. LS and AL genotyped Arabidopsis lines. PA performed imaging. AG and PA drafted the manuscript. All authors contributed to the article and approved the submitted version.


Phylogenomic Analysis of Salmonella enterica Serovar Indiana ST17, an Emerging Multidrug-Resistant Clone in China

ABSTRACT Salmonella enterica serovar Indiana (S. Indiana) is an extremely expanded foodborne pathogen in China in recent years. This study aimed to elucidate the national prevalence and phylogenomic characterization of this pathogen in China. Among 5, 287 serotyped Salmonella isolates collected during 2002 to 2018, 466 S. Indiana isolates were found in 15 provinces, and 407 were identified to be ST17, and the rest were ST2040. Among 407 ST17 isolates, 372 (91.4%) were multidrug resistant, and 366 (89.9%) were resistant to ciprofloxacin, 235 (57.7%) were further resistant to ceftriaxone. Phylogenomic analysis revealed that ST17 isolates were classified into four clades (I, II, III and IV), which appeared in international clonal dissemination. ST17 isolates from China fell into Clade IV with part of isolates from the United Kingdom, the United States, South Korea, and Thailand, suggesting their close genetic relationship. Mutations in quinolone resistance-determining regions (QRDR) of GyrA and ParC, and plasmid-mediated quinolone resistance (PMQR) genes aac(6′)-Ib-cr, oqxAB, and qnrS as well as extended spectrum β-lactamases (ESBL) genes blaCTX-M, blaOXA, and blaTEM in isolates from Clade IV were much higher than those from other three clades. Various blaCTX-M subtypes (blaCTX-M-65, blaCTX-M-55, blaCTX-M-27, blaCTX-M-14, and blaCTX-M-123) with ISEcp1, IS903B, ISVsa5, and IS1R were found in ST17 isolates, especially Tn1721 containing ΔISEcp1-blaCTX-M-27-IS903B in P1-like bacteriophage plasmids. These findings on the prevalent and genomic characterization for the S. Indiana multidrug-resistant ST17 clone in China, which have not been reported yet, provide valuable insights into the potential risk of this high-resistant clone. IMPORTANCE Fluoroquinolones and cephalosporins are the primary choices for severe salmonellosis treatment. S. Indiana has become one of the most prevalent serovars in breeding poultry and poultry meats in China in recent years. ST17 was recognized as the leading epidemiological importance in S. Indiana because of its high-level resistance to the most of common antibiotics, including ciprofloxacin and ceftriaxone. However, the prevalence and phylogenomic characterization of ST17 isolates are unclear. Here, we did a retrospective screening on a large scale for S. Indiana in China, and performed its phylogenomic analysis. It was found that ST17 isolates had extensive spread in 15 provinces of China and became a multidrug-resistant clone. The international spread of the ST17 isolates was observed among several countries, especially China, the United Kingdom, and the United States. Our study emphasized the importance of surveillance of a high-resistant S. Indiana ST17 clone to combat its threat to public health.

Fluoroquinolones and cephalosporins are often used as the first-line drugs for salmonellosis treatments upon the failure of other therapeutic options. Ciprofloxacin and ceftriaxone are the representative antibiotics of fluoroquinolones and cephalosporins classes, respectively. However, ciprofloxacin and ceftriaxone co-resistance S. Indiana isolates have emerged, and become prevalent in retail meats and patients (8,9). Furthermore, these isolates generally are multidrug-resistant (MDR) (6,9,14). We are now facing a formidable and growing menace from the emergence of MDR S. Indiana, and thus, it is vital to uncover epidemiological and evolutionary characterizations as well as antibiotic resistance mechanisms of this pathogen.
To date, some conventional methods such as PCR, pulse-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) have been used to explore the prevalence of S. Indiana (8,12,15). However, the conventional surveillance makes it difficult to obtain primary transmission dynamics of the expanded S. Indiana. Currently, whole-genome sequencing has been widely applied to reveal the evolutionary dynamics of bacteria (16)(17)(18). Therefore, a retrospective screening on a large scale was performed on 5, 287 serotyped Salmonella isolates from 16 provinces of China to elucidate the national prevalence of S. Indiana. The 171 genomes of S. Indiana isolates in this study together with those from other countries were used to give an insight into the phylogenomic structure and evolutionary characterizations of S. Indiana. Finally, the contribution of genetic factors to antibiotic resistance was explored in this pathogen.

RESULTS AND DISCUSSION
S. Indiana ST17 existed mainly in meats, patients, and healthy carriers in multiple provinces of China. A total of 466 (8.81%) S. Indiana isolates were obtained from 5, 287 serotyped Salmonella isolates from foods, patients, healthy carriers, and environments in 15 of 16 provinces (Shanghai, Shandong, Guangdong, Guangxi, Hubei, Chongqing, Shaanxi, Shanxi, Heilongjiang, Beijing, Henan, Hebei, Zhejiang, Fujian, and Xinjiang) (Fig. 1). Among 466 isolates, 407 (87.3%) were identified to be ST17, and the rest (12.7%) were ST2040. The detection positive rate (87.3%) of ST17 isolates was lower than that in a previous study (100.0%) (9), which might result from the emergence of ST2040 isolates. To our knowledge, S. Indiana ST2040 has not been reported before, and further research will be needed on its prevalence and biological characteristics.
In this study, food samples (64.4%; 262/407) were the predominant sources of ST17 isolates (Table S1). It was further demonstrated that food samples were mainly composed of chicken, pork, duck, and marine products. A total of 186 (45.7%) isolates were recovered from chicken, accounting for the largest portion, and then duck (9.8%) and pork (3.7%) (Table S1), which suggested that chickens were the main vehicle of S. Indiana ST17 isolates. In addition to foods, 13.5% of ST17 isolates were found in healthy carriers (Table S1), which could be explained by its lower pathogenicity compared to S. Enteritidis and S. Typhimurium (19). However, healthy carriers with S. Indiana could not receive prompt treatment because they are asymptomatic. This was likely to accelerate the spread of S. Indiana isolates among humans. Besides, 15.2% of ST17 isolates were recovered from patients with diarrhea, which were mostly children or the elderly with low immunity.
High-level multidrug resistances, including fluoroquinolones and cephalosporins resistance, occurred in S. Indiana ST17 from China. In this study, 366 (89.9%) of 407 S. Indiana ST17 isolates were resistant to ciprofloxacin, and 280 (68.8%) were resistant to ceftriaxone (Table S1) (Fig. 2). More importantly, a total of 372 (91.4%) ST17 isolates were identified to be MDR ones (Table S1). This high MDR rate of ST17 isolates was consistent with that (91.1%) in a previous study (9). Furthermore, 305 (74.9%) and 159 (39.1%) isolates were resistant, respectively, to at least five and seven classes of antibiotics. It was noted that an extensively drug-resistant S. Indiana ST17 isolate with resistance to 10 classes of antibiotics, including ciprofloxacin and ceftriaxone was obtained from a chicken carcass in 2012 in Guangdong, China in our previous study (20). Ciprofloxacin and ceftriaxone are the common drugs of choice for treating Salmonella infections (21). The high-level MDR in S. Indiana ST17 isolates, especially ciprofloxacin and ceftriaxone co-resistance would greatly increase the challenge of clinical treatments for patients.
Currently, S. Indiana isolates in other countries were relatively susceptible to most of the common antibiotics (22,23). For example, S. Indiana isolates from Ethiopia exhibited susceptibility to ampicillin, amoxicillin-clavulanic acid, chloramphenicol, cephalothin, cefoxitin, streptomycin, and sulfamethoxazole-trimethoprim (22). Compared to those from other countries, it appeared that the high-level antibiotic resistance in S. Indiana was a unique phenomenon in China, which was likely to be caused by the widespread use of antibiotics in China. In 2013, the total amount of antibiotics used in China was about 162, 000 tons, approximately 160 times that of the United Kingdom, and 48% of which was used for human consumption, and the rest was shared by animals (24). Furthermore, the production yield of fluoroquinolones (including ciprofloxacin) and b-lactams (including ceftriaxone) in China was estimated to be 27, 300 and 34, 100 tons, respectively (24). Therefore, governmental regulations limiting the use of antimicrobial agents have been issued in China to reduce the potential threat of MDR bacteria to public health. International spread occurred in S. Indiana ST17 isolates. To further determine the phylogenetic characteristics of S. Indiana isolates, phylogenomic analysis was performed on 171 genomes from China in this study and 470 genomes in the public databases from other 12 countries. A total of 3, 668 core SNPs were extracted from these 641 S. Indiana genome sequences, 2, 807 core SNPs of which were used to construct a maximum likelihood tree after 861 core SNPs in the recombinant regions were removed.
A total of six sequence types (STs) (ST17, ST2040, ST3480, ST3558, ST5311, and ST5449) were identified in these 641 S. Indiana isolates, of which ST17 (n = 594) and ST2040 (n = 39) were the major types (Fig. 3a). Compared to ST2040 isolates, ST17 isolates were widely disseminated in 13 countries tested across Europe, North America, and Southeast Asia, and persisted for many years (1970 to 2020) ( Fig. 3b and Fig. S1). Furthermore, it was found in Fig. 3b that ST17 and ST2040 clones had emerged, which could be proved by their long genetic distance.
Phylogenomic results indicated ST17 isolates might early emerge in the United States because they were the base of the phylogenetic tree, and then were evolved in Ireland and the United Kingdom, and finally separated into four clades (I, II, III, and IV) internationally (Fig. 3b). Clade I was composed of 250 isolates from the United Kingdom between 2014 and 2020. Clade II was a mixed cluster, which was composed of isolates from the Netherlands, Germany, and the United Kingdom in European, suggesting that their genetic relationships were close. Clade III was composed of isolates from the United States and the United Kingdom, and their evolutionary path implied that a major introduction might occur in the United States from the United Kingdom, and then nationwide spread. Furthermore, ST17 isolates from China during 2006 to 2018 fell into Clade IV, and four human isolates from the United Kingdom also belonged to Clade IV ( Fig. 3b and Fig. S2). It was quite possible that the persons traveled to China, returned to the United Kingdom, and then became sick. Other sources of isolates from different countries were found in Clade IV including chicken (South Korea, n = 1), porcini Mushrooms (United States, n = 1), and dried chill (Thailand, n = 1) (Fig. S2), suggesting their close genetic relationship to those from China.
Mutations in GyrA and ParC as well as PMQR genes frequently happened in S. Indiana ST17 from China. It was observed in isolates from China that mutations in GyrA-ParC (84.8%), PMQR genes of aac(69)-Ib-cr (62.0%) and oqxAB (29.8%) were frequently identified. Mutations in QRDR of GyrA were commonly found as S83F/D87N (57.3%) and S83F/D87G (24.6%), followed by S83F (1.8%) and D87G (1.2%). Mutations in ParC were commonly found as T57S/S80R (n = 83.0%), followed by T57S (17.0%). Compared with S. Enteritidis and S. Typhimurium (25,26), S. Indiana isolates possessed more site mutations in GyrA and ParC. Furthermore, it was found that rates of mutations in GyrA and ParC in S. Indiana isolates from China were significantly higher (P , 0.05) than those from the United Kingdom, United States, and Germany (Table S3). The mutations in GyrA-ParC were identified in 145 (84.8%) isolates from China, but these mutations were only found in 12 (3.7%) and 2 (2.4%) isolates from the United Kingdom and the United States, respectively (Table S3). In addition, the detection positive rate of aac(69)-Ib-cr and oqxAB in S. Indiana isolates from China were significantly higher (P , 0.05) than those from the United Kingdom, United States, and Germany. Mutations in QRDR and PMQR genes were mainly responsible for the decreased susceptibility to ciprofloxacin in Gram-negative bacteria (27). These results showed that mutations in GyrA and ParC as well as PMQR genes were frequently identified in S. Indiana ST17 from China, which could be a reason for the high-level ciprofloxacin resistance in this study.
It was found in Fig. 3b that a mutation also occurred in ParC (T57S) in ST2040 isolates. It was interesting that the mutation in ParC (T57S) was identified in all 641 S. Indiana isolates tested including ST17 and ST2040 isolates in this study from 13 countries across the years of 1970 to 2020, suggesting that there might be a natural mutation site in S. Indiana.
High-level MICs of ciprofloxacin against ST17 isolates resulted from the combination of mutations in GyrA-ParC and PMQR genes. As shown in Fig. 4, there were multiple site mutations in QRDR accompanied by a dramatic clonal expansion in ST17 isolates in China through analysis of a reconstructed phylogenetic tree. In particular, a single mutation occurred in GyrA (S83F or D87N) in the base isolates of the ST17 clone, followed by double mutations in GyrA (S83F/D87N or S83F/D87G) with an additional mutation in ParC (S80R). Similarly, phylogenomic analysis indicated that the early mutation occurred in GyrA (S83F), followed by a mutation in ParC (S80I) with a dramatic expansion in Salmonella Kentucky ST198 (28). These findings implied that the increase in site mutations in GyrA and ParC might contribute to the expanded dissemination of S. Indiana ST17 through enhancing its antibiotic resistance.
The mutations in GyrA and ParC were constructed in a susceptible wild-type isolate (S. Indiana SJTUF14139) through homologous recombination yielding mutants of PML1 (GyrAS83F), PML2 (GyrAD87N), PML3 (GyrAS83F/D87N), and PML4 (GyrAS83F/D87N-ParCS80R) to examine their role in ciprofloxacin resistance formation (Table S6). The results from growth curves showed that mutations in GyrA and ParC had no significant effect on the growth rate of PML1-4 (Fig. S4). Furthermore, a single mutation in GyrA (S83F or D87N) or double mutations in GyrA (S83F/D87N) resulted in an increase in MIC of ciprofloxacin from 0.015 mg/mL to 0.25 mg/mL. Triple mutations (GyrAS83F/D87N-ParCS80R) in PML4 resulted in a higher MIC of ciprofloxacin (8 mg/mL), which exceeded the breakpoint resistance value (1 mg/mL) according to the CLSI 2019 profile.
Our previous study indicated that the MICs of ciprofloxacin against S. Indiana were generally higher than 64 mg/mL (15), and it seemed that mutations in GyrA and ParC were not the only reason for the high-level MIC of ciprofloxacin. Therefore, a recombinant plasmid pMDoqxAB was constructed by cloning PMQR gene oqxAB into pMD19-T vector, which was then introduced into PML1-4 to yield the mutants PML5 (GyrAS83F-pMDoqxAB), PML6 (GyrAD87N-pMDoqxAB), PML7 (GyrAS83F/D87N-pMDoqxAB) and PML8 (GyrAS83F/D87N-ParCS80R-pMDoqxAB). Compared with the wild-type strain SJTUF14139, the growth rates of PML5, PML6, PML7, and PML8 were lower (Fig. S4), which might result from fitness cost through the introduction of pMDoqxAB. Compared with those in PML1-4, the MICs of ciprofloxacin against PML5-8 were increased by 4 to 8 times. The MICs of ciprofloxacin against PML5, PML6, and PML7 reached at least 1 mg/mL, which indicated that mutations in GyrA (S83F and/or D87N) combined with oqxAB could make the MIC of ciprofloxacin reach the breaking resistance point (1 mg/mL). Moreover, the MIC of ciprofloxacin against PML8 (GyrAS83F/D87N-ParCS80R-pMDoqxAB) reached 32 mg/mL, which was 32 times higher than the breaking resistance point (1 mg/mL). These findings indicated that highlevel MICs of ciprofloxacin against ST17 isolates resulted from the combined action of mutations in GyrA-ParC and PMQR genes.
Multiple antibiotic resistance genes (ARGs) appeared on IncHI2-IncHI2A plasmids in ST17 isolates. It was found that there were diverse types of plasmids in ST17 isolates, which were IncHI2-IncHI2A, IncN, IncQ1, IncQ2 and P1 phage-like types. In this study, the IncHI2-IncHI2A plasmid was the most frequently observed in S. Indiana isolates in China, accounting for 68.4%. Therefore, an IncHI2-IncHI2A plasmid (p14154A) was extracted from the SJTUF14154 isolate and further sequenced for analysis. It was found that p14154A was highly similar to our previous reported plasmid p87912 (Accession number CP041180; 100% coverage; 100% identity) (20) and p13520 (Accession number CP041182; 100% coverage; 99.2% identity) (15) (Fig. 5a). The hosts of these three plasmids were from different places (p14154A, Shandong; p13520, Shanghai; p87912, Guangdong), suggesting that these plasmids were likely to evolve from the same ancestor. A total of 26 ARGs were found to be located in the multidrug resistance region (MRR) with a size of ;75 kbp in plasmid p14154A. In our previous study, an IncHI2-IncHI2A plasmid p87912 was transferred from the donor to the recipient E. coli DH5a accompanied by the transfer of MDR phenotype (20), which indicated that IncHI2-IncHI2A plasmids bearing multiple ARGs played an important role in MDR in S. Indiana.
It was found that ARGs of bla CTX-M-65 , mphA, qepA, oqxAB, bla OXA-1 , fosA3, and rmtB interspersed with different ISs and transposons (Tns) (IS26, ISEcp1, ISCR3, ISVsa3, IS1006, IS903B, Tn1721, and Tn3-like) in plasmid p14154A. The genomic rearrangements and genetic exchanges were always mediated by mobile elements (29). For example, IS26 is the common mobile element, which is the major component of antibiotic resistance determinants. Replicative transposition or homologous recombination on conservative regions frequently occurred in IS26, and then created a new MRR (29,30). The current study and previous studies (15,20,30) showed that most of MRRs had a mosaic structure bound at both ends by fragments of mobile elements, which implied that the formation of an MRR in S. Indiana ST17 was involved in the acquisition and stepwise integration of multiple ARGs determinants through homologous recombination or transposition of mobile elements. P1-like bacteriophage plasmids bearing Tn1721-bla CTX-M-27 emerged in ST17 isolates. In this study, nine ST17 isolates were identified to possess P1-like replicon genes, and six were found to be positive for ESBL gene bla CTX-M-27 , which was seldomly found in other isolates. Therefore, a plasmid p14146 was extracted from the SJTUF14146 isolate and sequenced for analysis, which was proved to be a P1-like bacteriophage plasmid, sharing the high similar sequence structure with Enterobacteria phage P1 (Fig. 5b). Furthermore, a Tn1721 unit harboring bla CTX-M-27 was integrated into p14146, and it was flanked by two 6 bp direct repeat sequences (TATGAA), suggesting a transposition event occurred (Fig. 5c). Gene bla CTX-M-27 in the Tn1721 unit was linked to the transposable element DISEcp1 upstream and IS903B downstream. Our study and previous studies (31,32) indicated that bla CTX-M-27 was frequently located in Tn1721 transposon unit in S. Indiana ST17, which implied that Tn1721 might accelerate the horizontal transfer of bla CTX-M-27 gene through transposition.
Multiple bla CTX-M variants existed in ST17 isolates from China. It was demonstrated in Table S3 that ESBL genes bla OXA-1 , bla CTX-M and bla TEM-1B were identified, respectively, in 106, 89 and 36 isolates from China (Table S3) (35). In particular, these bla CTX-M genes were generally located on the plasmids, which might contribute to the rapid expansion of ceftriaxone resistance through conjugation among Enterobacteriaceae (36)(37)(38).
As shown in Fig. 5c, gene bla CTX-M-65 was frequently linked to the transposable elements DISEcp1 upstream and IS903B downstream, and this transposon unit was about 4.9 kbp. It was found in p14154A that the truncated DISEcp1 upstream of bla CTX-M-65 was interrupted by IS26. On the contrary, a complete ISEcp1 element was found in the transposon units of bla CTX-M-14 and bla CTX-M-123 , inserting the upstream of a complete element ISVsa5 and IS1R. The difference in the transposon unit between bla CTX-M-14 and bla CTX-M-123 was that downstream of the former was IS903B, but that of the latter was Dorf477. Gene bla CTX-M-55 was frequently found to be linked to ISEcp1 upstream and Dorf477 downstream. The horizontal transfer of ARGs is found to be mediated by mobile genetic elements like transposons, insertion sequences, and integrons (39). Insertion sequences are able to move themselves and ARGs to new locations in the same or different DNA molecules such as chromosomes and plasmids (29). A previous study demonstrated that ISEcp1 could move bla CTX-M-2 from the chromosome to the plasmid in Kluyvera ascorbata through transposition (40). The current study showed that ISEcp1 was the most common mobile element associated with bla CTX-M genes, which might play a key role in the horizontal transfer of bla CTX-M genes in S. Indiana.
Conclusion. Characterization on the large-scale emergence and international spread of an S. Indiana ST17 clone was systematically carried out in this study, which has not been reported before. High-level MDR including ciprofloxacin and ceftriaxone resistance was found in the ST17 clone from China, which might be explained by their high mutations in GyrA and ParC as well as multiple ARGs, including PMQR genes and ESBL genes from plasmids. Our study suggests that necessary strategies are warranted to prevent the further dissemination of this high-risk MDR ST17 clone. Therefore, China government has issued regulations to prohibit excessive use of antibiotics in livestock farms and hospitals, and to strengthen education and publicity on bacterial resistance.

MATERIALS AND METHODS
Bacterial isolates. A total of 5, 287 Salmonella isolates from foods, patients, and environments in 16 provinces of China were collected between 2002 and 2018. Food sources included chicken, pork, duck, marine products, and frozen food. Human sources included the stool and blood of health checkers, outpatients, and inpatients in hospitals for diarrhea treatment. Environment sources included municipal sewage and poultry feces. All above Salmonella isolates were identified via API20E test strips (bioMérieux, France) and serotyped via commercial antiserum (Statens Serum Institute, Copenhagen, Denmark) according to the manufacturer's guidelines.
Antimicrobial susceptibility testing. Antimicrobial susceptibility testing was performed on S. Indiana isolates using the agar dilution method provided by the Clinical and Laboratory Standard Institute (41)  Whole-genome sequencing. Bacterial cells were grown in 250 mL liquid Luria-Bertani (LB) medium overnight at 37°C, with agitation at 200 rpm. Genomic DNA was extracted from S. Indiana isolates using the QIAamp DNA minikit (Qiagen, CA). The sequencing service was provided by the Personal Biotechnology Company (Shanghai, China). The whole-genome sequencing (WGS) was performed by using the Illumina MiSeq platform (Illumina, San Diego, CA, USA). The plasmid sequences were extracted from the whole-genome sequences using the PacBio RS II system (Pacific Biosciences, Menlo Park, CA, USA). For the Illumina MiSeq platform, a 400-bp DNA library was constructed and sequenced by the paired-end sequencing mode. Data generated from the Illumina MiSeq platform was assembled by SPAdes (43). For the PacBio RS II platform, a 10-kbp DNA library was constructed and sequenced using single-molecule real-time (SMRT) sequencing technology. Sequence data from the PacBio RS II platform was assembled using the Canu software (44). Finally, the consensus genome sequence was determined using the Pilon software (45). Sequence data of read numbers, total bases, GC contents, and Q20/30 rates was available in Table S7 in the supplemental material.
Antimicrobial resistance determinants. ResFinder 4.1 was used to identify ARGs and chromosomal mutations mediating antibiotic resistance in the genome (49). MLST 2.0 was used to identify the STs of bacteria (50). Plasmidfinder was used to identify replicon types of plasmids (51). ISfinder (https://www-is .biotoul.fr/) was used to analyze the IS and transposons in the genome.
Phylogenomic analysis. A total of 171 genomes in this study, including a reference genome of S. Indiana SJTUF87912v2 (Accession number CP041179.1) with 470 genomes of S. Indiana isolates available from the Enterobase database (http://enterobase.warwick.ac.uk/species/index/senterica, August 2021) were used for phylogenomic analysis. Single-nucleotide polymorphisms (SNPs) were extracted using Snippy (https://github.com/tseemann/snippy) to generate core genomic alignment. Gubbins (52) was then used to remove recombination regions. The core SNP alignment was used to generate a maximum-likelihood phylogeny using RAxML v8.1.23 (53) with the GTR nucleotide substitution model. Furthermore, 100 random bootstrap replicates were conducted to assess the node support. The phylogenetic tree was visualized together with metadata using Microreact v5.99.0 (54).
Gene cloning and generation of gyrA and parC mutants. The oqxAB, gyrA and parC fragments were amplified by PCR using primers (Table S4), and then cloned into pMD19-T vector, yielding recombinant plasmids pMD19-T-oqxAB and pMD19-T-gyrA/parC. Recombinant plasmid pMD19-T-oqxAB was transformed into E. coli DH5a using electroporation. A suicidal vector pRE112 was used to construct gyrA and parC mutants through homologous recombination (55). Point mutations on plasmids pMD19-T-gyrA/parC were constructed using QuikChange Lightning Site-Directed Mutagenesis kit (Agilent, USA). The above-constructed plasmids were then ligated to pRE112, yielding recombinant pRE112-gyrA/parC plasmids. Then, the obtained pRE112-gyrA/parC recombinant plasmids were transformed into an S. Indiana wild-type strain SJTUF14139 with a pKOBEG plasmid in advance. Along with inducing by arabinose and saccharose, wild gyrA/parC sequence bases were replaced with point mutations through homologous recombination. The amino acid substitutions of GyrA and ParC were analyzed and aligned using BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM= blastp&PAGE_TYPE=BlastSearch&BLAST_SPEC=&LINK_LOC=blasttab&LAST_PAGE=blastn) to confirm the specific mutations. The above plasmids used in this study for constructing mutants could be found in Table S5 in the supplemental material.
Growth curves. The wild-type strain SJTUF14139 and its mutants were inoculated into 30 mL fresh LB broth overnight at 37°C and 200 rpm. Then, bacterial suspensions were adjusted to an OD 600 nm of approximately 0.1. The growth curves of bacteria were measured at regular time intervals (1 h) by a Bioscreen C Analyzer (TYPE FP-1100-C, Oy Growth Curves Ab Ltd.). Experiments were performed in three independent assays.
Data availability. The genome sequence data were available from BioProject ID: PRJNA641453, and a reference genome SJTUF87912v2 was deposited in the NCBI database under the accession number


PCARE requires coiled coil, RP62 kinase-binding and EVH1 domain-binding motifs for ciliary expansion

Abstract Retinitis pigmentosa (RP) is a genetically heterogeneous form of inherited retinal disease that leads to progressive visual impairment. One genetic subtype of RP, RP54, has been linked to mutations in PCARE (photoreceptor cilium actin regulator). We have recently shown that PCARE recruits WASF3 to the tip of a primary cilium, and thereby activates an Arp2/3 complex which results in the remodeling of actin filaments that drives the expansion of the ciliary tip membrane. On the basis of these findings, and the lack of proper photoreceptor development in mice lacking Pcare, we postulated that PCARE plays an important role in photoreceptor outer segment disk formation. In this study, we aimed to decipher the relationship between predicted structural and function amino acid motifs within PCARE and its function. Our results show that PCARE contains a predicted helical coiled coil domain together with evolutionary conserved binding sites for photoreceptor kinase MAK (type RP62), as well as EVH1 domain-binding linear motifs. Upon deletion of the helical domain, PCARE failed to localize to the cilia. Furthermore, upon deletion of the EVH1 domain-binding motifs separately or together, co-expression of mutant protein with WASF3 resulted in smaller ciliary tip membrane expansions. Finally, inactivation of the lipid modification on the cysteine residue at amino acid position 3 also caused a moderate decrease in the sizes of ciliary tip expansions. Taken together, our data illustrate the importance of amino acid motifs and domains within PCARE in fulfilling its physiological function.

Introduction
The vertebrate retina captures, amplifies and propagates light signals using intricately connected layers of photoreceptors, retinal neurons and optic nerve cells. This mechanism relies on the ability of photoreceptors to capture light photons and convert them into electrochemical signals via the action of opsin proteins that populate the stacked, disk-shaped membranes of photoreceptor outer segments (OSs). Photoreception and transduction place these OS under high oxidative stress, and therefore damaged components of the membrane disks require constitutive renewal of the OS. This is achieved by shedding of the OS tips at the rate of ∼10% per day, which is matched by OS biogenesis at the base (1). To support the highly metabolically active OS, a photoreceptor contains a biosynthetic inner segment that is connected to the OS by a microtubule-based bridge called the connecting cilium that is largely homologous to the transition zone of a primary non-motile cilium (2,3). The connecting cilium enters the OS in the region that cradles new opsin-loaded disks, which are formed by evagination and expansion of the ciliary plasma membrane (4,5). A branched F-actin network localizes at the site of evagination; however, an exact mechanism of its involvement in disk morphogenesis remains elusive (6). Mutations in PCARE (photoreceptor cilium actin regulator) (C2orf71) have been linked to autosomal recessive retinitis pigmentosa type 54 (RP54) (7,8). PCARE has been found to localize to the evagination sites of photoreceptors where it interacts with WASF3 (WAVE3, SCAR3) protein (9). WASF3 is an actin modifier that is able to activate the Arp2/3 complex and facilitate assembly of a branched F-actin network (10,11). PCARE expression is specific to the retina; however, when PCARE is recombinantly expressed in cultured ciliated cells, it also localizes specifically to the cilium. Moreover, upon co-expression of WASF3, ciliary tip expansions appear that harbour both PCARE and WASF3 proteins. The formation of ciliary tip expansions is inhibited by F-actin poisons and siRNA-mediated downregulation of Arp2 expression (9).
This study aims to further investigate the role of PCARE in the actin dynamics of the ciliary tip by scrutinizing the structure of this novel protein. PCARE is an intrinsically disordered protein, which limits attempts to determine its molecular structure using crystallographic methods (12). However, advances in the in silico predictions of protein structures allow us a glimpse into its structural and functional motifs. Previously, we described a tentative actin-binding WH2 short linear motif (SLiM) between amino acids 597 and 615 (9). WH2 is one of several SLiMs found in proteins that are involved in the regulation of actin filament assembly (13). However, when cotransfected with WASF3 in hTERT RPE-1 and IMCD-3 cell lines, PCARE lacking this WH2 motif retained the ability to form ciliary tip expansions (data not shown). This finding, together with an absence of robust motif representation in the eukaryotic linear motif (ELM) database (13) and low conservation score, prompted us to further study the structure-function relationship within the PCARE protein. We designed a bioinformatic study to predict evolutionary conserved amino acid motifs of PCARE. Subsequently, we revealed important functional amino acid motifs by expressing the PCARE mutant constructs lacking the motifs in hTERT RPE-1 and IMCD-3 cell lines and assessing their ability to localize to the cilia and form ciliary tip expansions. This work thereby points to a potential mechanism of actin dynamics during photoreceptor disc formation in health and disease.

Bioinformatics analysis of PCARE protein showed structural and amino acid motifs potentially involved in interaction with actin and proteins involved in actin remodelling
PCARE is a retina-specific protein consisting of 1289 amino acids with a predicted molecular weight of 140 kDa. On the basis of the prediction using the DISOPRED and AlphaFold servers, the PCARE protein is mostly intrinsically disordered with a likely structured region between amino acids 171 and 338 (14,15). This region corresponds to a helical domain framed with two long helices forming an antiparallel coiled coil predicted with both the PSIPRED workbench and AlphaFold (16). The amino acid residue analyses showed no other strongly predicted structural domains (Supplementary Material, Fig. S1). Bioinformatic analysis revealed that the glycine residue at amino acid position 2 (p.Gly2) and the cysteine residue at amino acid position 3 (p.Cys3) may undergo myristoylation and palmitoylation lipid modifications, respectively (7). In the middle part of the protein, two target sites for the MAK (RP62) kinase were predicted (amino acid positions 513-516 and 577-580) and, in a proline-rich region, three enabled/VASP homology 1 domain (EVH1)-binding motifs (amino acids positions 805-809, 830-834 and 1056-1060).

PCARE mutants show stable protein expression comparable with wild-type
We modified recombinant PCARE cDNA to express a series of HA-tagged PCARE protein variants containing disrupted amino acid motifs (Fig. 1). To prevent lipid modification on the second glycine and third cysteine residues, the cDNA sequence was changed to encode the catalytically inert amino acid residue alanine at these positions. All other predicted amino acid motifs were disrupted by deleting the entire motif. We observed that the mutant PCARE constructs were stably expressed when transfected into HEK293T cells and their levels of expression were comparable to that of the recombinant wildtype protein (Fig. 2).

Disturbance of predicted PCARE motifs differentially affects ciliary characteristics
When transfected into hTERT RPE-1 cells, wild-type PCARE has been shown to localize along the cilia (9). We repeated this experiment with wild-type and mutant PCARE isoforms and observed ciliary localization for each of them except for the mutant harbouring the helix domain deletion (PCARE helix) where the protein localized into the cytosol only (Fig. 3).
When co-transfected with WASF3, PCARE induces the formation of ciliary tip expansions. We classified three ciliary phenotypes: type 1, where PCARE and WASF3 co-localize into the cilia and form expansions of the ciliary tip; type 2, where the proteins co-localize to the cilia but form no expansions and type 3, where cilia also appear normal but PCARE and WASF3 retain within the cytosol and/or nucleus (Fig. 4, Supplementary Material, Fig. S3). With wild-type PCARE, 50% of the colocalization events resulted in the formation of ciliary expansions of type 1 per total double-positive cilia (n = 271 number of double-positive cilia counted, SD = 7.8%). Mutant constructs formed less of type 1 expansions, for example, PCARE EVH1a, b and c formed 39%, 37% and 48% of type 1 expansions, respectively (n = 71, SD = 8.8%, n = 70, SD = 8.5% and n = 62, SD = 10.5%), whereas PCARE RP62a and PCARE RP62b (n = 71, SD = 10.0% and n = 66, SD = 9.6%) formed 48% and 42% of expansion, respectively ( Fig. 4A-C). We observed the second most prominent decrease in type 1 expansions in PCARE EVH1a-c (14%, n = 61, SD = 5.2%). PCARE helix (n = 34) failed to localize into the cilia and thus did not form expansions at all. Furthermore, we quantified the size of the ciliary tip expansions formed upon co-transfection of PCARE mutant constructs with WASF3 in hTERT RPE-1 cells. We observed the mean expansion size of wild-type PCARE to correlate with previous observations (0.97 μm 2 , n = 117, SD = 0.4 μm 2 ) (9). Our results showed that most mutated constructs formed smaller expansions than the wildtype. From those, the PCARE EVH1a and b constructs formed significantly smaller expansions with means at 0.82 μm 2 and 0.75 μm 2 , respectively (n = 73, SD = 19 μm 2 , P = 0.001 and n = 37, SD = 0.2 μm 2 , P < 0.0001). The tripledeletion PCARE EVH1a-c showed the smallest mean expansion size of 0.71 μm 2 (n = 10, SD = 0.22 μm 2 , P = 0.006). Also, a removal of lipid modification from p.Cys3 might result in a decreased expansion size (size = 0.84 μm 2 , n = 36, SD = 0.28 μm 2 , P = 0.035) (Fig. 4D). At the N-terminus of PCARE, the glycine residue at amino acid position 2 (p.Gly2), and the cysteine residue at amino acid position 3 (p.Cys3) exhibit tentative palmitoylation and myristoylation lipid modifications, respectively. As predicted with DISOPRED server, a helical structural domain is located between amino acids 171 and 338. Using Jalview, the following conserved SLiMs were predicted: two RP62 kinase-binding motifs, and three EVH1 domainbinding motifs. To generate PCARE mutant proteins, the p.Gly2 and p.Cys3 residues were substituted by the inactive amino acid alanine residue, whereas predicted protein domains that are annotated in this schematic were deleted via site-directed mutagenesis, including one mutant in which all three EVH1 domain-binding motifs were deleted.
The absence of lipid modification on p.Gly2 or deletions of RP62 candidate phosphomotifs did not exhibit a statistically significant difference in ciliary expansion size ( Table 1).

Transfection of PCARE mutant constructs into stable IMCD-3 expressing WASF3 confirms decreased ability to form tip expansions
To minimize the variability of the double-transfection experimental design, we also generated IMCD-3 cell lines stably expressing WASF3 that allowed for a standardized level of WASF3 expression between samples (Supplementary Material, Fig. S2). In the IMCD-3 cells, the average percentage of cilia with type 1 phenotype increased by 12% while type 3 decreased by 9%, whereas the average SD of expansion sizes decreased by 16%. Most of the results observed in IMCD-3 cells stably expressing WASF3 followed the trends of hTERT RPE1 cell results (Fig. 5). For example, removal of lipid modification from the cysteine residue at amino acid position 3 also showed a decrease inthe ciliary tip expansion sizes Figure 2. PCARE mutants are expressed and stable. Wild-type and mutant PCARE constructs were transfected in HEK293T cells. Western blot analysis revealed stable expression of all PCARE constructs. Wild-type PCARE protein has a molecular weight of 140 kDa, the molecular weight of mutant PCARE proteins is approximately the same, except for the mutant carrying the helix deletion which is smaller. Tubulin was loaded for control of the total amount of protein. In contrast to hTERT-RPE1, no effect on mean expansion size was observed for PCARE EVH1b (0.77 μm 2 , SD = 0.24 μm 2 , P = 0.052), but deletion of each MAK/RP62 When counted in the IHC slide and quantified, with wild-type PCARE normalized to hundred percent, all PCARE mutants generated less ciliary tip expansions than the wild-type (type 1 in yellow, type 2 in green, type 3 in red). (B) hTERT RPE-1 cells were transfected with 3×HA-PCARE (wild-type or mutant) and 3×FLAG-WASF3 constructs in equal amounts. Three distinct types of the ciliary tip expansions can be distinguished: type 1, where PCARE and WASF3 co-localize into the cilia and form expansions of the ciliary tip, type 2, where the proteins co-localize to the cilia but formed no expansions and, finally, type 3, where the proteins are retained in the cytosol (3×HA-PCARE in green, WASF3 in red, ARL13B in magenta, DAPI in blue). Untransfected cells (Utr) are shown as a negative control. Scale bars 5 μm. (C) The sizes of the ciliary tip expansions of type 1 were counted and quantified using an automated Fiji script. The triple-deletion PCARE EVH1a-c and single-deletions of PCARE EVH1a and PCARE EVH1b showed a significant decrease in the size of the expansion tip ( * * * P ≤ 0.001, * * P ≤ 0.01, * P ≤ 0.05, red line shows mean and SD). Furthermore, for PCARE EVH1c, a decrease of expansion size was also observed (mean = 0.75 μm 2 , n = 115, SD = 0.26, P = 0.014) ( Table 1).

Discussion
Mutations in PCARE are causative for retinal ciliopathy RP54 (7,8). Recently, we observed that PCARE localizes at the initiation site of new OS disks within photoreceptors and proposed its role in protein transport and actin remodelling that is required to form OS disks (9). We identified candidate SLiM motifs of PCARE that may be relevant for interactions with actin or actin modifiers and generated stable PCARE protein mutants with deletion or inactivation of these motifs. Our data strongly suggest the importance of the alpha-helix domain for PCARE ciliary trafficking and EVH1 domain-binding motifs and myristoylation on the third cysteine for modification of the actin filament network in the ciliary tip. The aim of this study was to investigate the structurefunction relationship within PCARE with regard to its ability to regulate actin dynamics. To address this, we performed a bioinformatics analysis of PCARE protein structure to find evolutionary conserved structural and functional amino acid motifs. From the list of predicted SLiMs, we selected the motifs that are potentially involved in interaction with actin or with other proteins involved in actin remodelling. We identified two target sites for a photoreceptor ciliumassociated MAK/RP62 that phosphorylates serine and threonine residues (17). Dysfunction of MAK kinase is linked to structural defects in photoreceptor cilia and recessive retinitis pigmentosa (18)(19)(20). MAK knockout mice retina shows elongated cilia, rhodopsin accumulation and photoreceptor degeneration (21). Furthermore, we identified three EVH1 domain-binding SLiMs that are specific to proteins that are involved in actin-based cytoskeleton reorganization, actin dynamics modulation and actin motility (22)(23)(24). We also modified amino acids (p.Gly2 and p.Cys3) at the N-terminus of PCARE, because of their known role as a lipid modification sites in transport and membrane localization of proteins (17). Finally, PCARE contains one predicted alpha-helical domain, one of the most common secondary structure motifs found within proteins (18). To access the function of each of these motifs, we generated PCARE mutant constructs with deletion or inactivation of the motifs of interest on the basis of wild-type PCARE cDNA.
To determine the inf luence of the predicted amino acid motifs on the PCARE stability, we expressed the PCARE mutant constructs in HEK293T cells and assessed PCARE expression levels. All mutant proteins were expressed at levels comparable to wild-type PCARE. Furthermore, to elucidate the function of the selected motifs in PCARE ciliary trafficking, we investigated the localization pattern of PCARE mutants in ciliated hTERT RPE-1 and IMCD-3 cells. Previously, we showed that, when separated into arbitrarily chosen fragments, the most Nterminal fragment of PCARE, which harbours the alphahelical coiled coil domain, localizes to the basal body and axoneme of the cilia and is sufficient to transport WASF3 into the cilia to form ciliary tip expansions (9). Interestingly, the most C-terminal fragment of PCARE alone was also capable of localization into the ciliary axoneme.
Here, we showed that full-size PCARE lacking the helix domain failed to localize to the cilia. This finding hints towards a crucial role that the minimal structural organization of PCARE has in its ciliary localization and further work is required to establish the exact role of the coiled coil domain in PCARE trafficking. Moreover, myristoylation and palmitoylation were predicted at the N-terminus of PCARE, modifications that are known to play a role in ciliary trafficking in Trypanosoma brucei and in eukaryotic cells (19,20). N-myristoylation on p.Gly2 is a protein modification where 14-carbon saturated fatty acid myristate is covalently attached to the N-terminal Gly (exposed after removal of p.Met1) of the target protein catalyzed by N-terminal myristoyltransferases. On p.Cys3, palmitoylation where 16-carbon fatty acid palmitate is covalently attached to a cysteine residue is predicted, a process that is catalyzed by the Nterminal palmitoyl acyltransferases. Myristoylation at the N-terminus also facilitates palmitoylation (25,26). The lipid-modified protein is directed to a membrane by the action of N-myristoylation, but palmitoylation is required for stable anchoring to the phospholipid bilayer (27). In X-linked retinitis pigmentosa protein 2 (RP2), myristoylation alone was not sufficient to traffic amino acid residues 1 to 16 RP2 to the cilia (20). We deactivated palmitoylation on Gly2 or myristoylation on Cys3 by Gly-to-Ala or Cys-to-Ala substitutions, but observed no effect on the ciliary trafficking. Further analysis of human retinal-organoid derived, or murine purified PCARE protein, for example by mass spectrometry, would be required to determine if p.Gly2 or p.Cys3 is respectively myristoylated or palmitoylated in vivo. Taken together, our data show that all PCARE mutants except for PCARE helix localized to the cilia, suggesting redundancy or potentially undiscovered amino acid motifs to play a role in ciliary trafficking of PCARE.
Next, we accessed the frequency and size of ciliary tip expansions generated by PCARE mutant proteins when co-expressed with WASF3, either via double transfection of both proteins in hTERT RPE-1 cells, or by a single transfection of PCARE in IMCD-3 cells stably expressing WASF3. All mutants showed a decreased frequency of ciliary tip expansions compared with the wild-type PCARE. In IMCD-3 cells stably expressing WASF3, there is a general increase in the ability to form ciliary tip expansions for all protein variants compared with double-transfected hTERT RPE-1. Compared with the wild-type, both PCARE (p.Gly2Ala), where the myristoylation was inactivated and PCARE (p.Cys3Ala), where the palmitoylation was inactivated, showed a lower number of ciliary tip expansions of type 1. When quantifying the sizes of the ciliary tip expansions, the deactivation of myristoylation had no statistically significant effect but the deactivation of palmitoylation seemed to have negatively affected the ability of PCARE to form ciliary tip expansions. Potentially, the palmitoylation-affected mutant impaired membrane anchoring and, thus, fewer units of the protein complex may reach the ciliary tip leading to fewer and smaller bulges.
Deletion of the EVH1 domain-binding motifs, both sequentially and concurrently, also resulted in fewer and smaller ciliary tip expansions in both hTERT RPE-1 and IMCD-3 cell lines. Analysis of the sequential deletions showed that the deletion of the most evolutionary conserved EVH1b motif affects the tip expansions most strongly in both cell lines. Analysis of concurrent EVH1 domain-binding SLiM deletions showed the strongest effect when all three EVH1 domain-binding motifs were deleted simultaneously. Although this effect is stronger than that of the individual deletions, more research should be done to explore a potential synergistic effect of the EVH1 deletions. Putative EVH1 domain-binding motifs would allow PCARE to interact with proteins that have the EVH1 (WH1, RanBP1-WASP; Pfam:PF00568) domain that is present in species ranging from yeast to mammals. Such proteins are involved in a wide range of signalling, nuclear transport and cytoskeletal events. Many EVH1-containing proteins are actin interactors and participate in actin cytoskeleton organization. The EVH1 domain-binding motifs of PCARE consist of five amino acid stretches containing three or more proline residues and resemble the EVH1 domain-binding motifs in other proteins such as the Ena/VASP family proteins, which are thought to assist in actin filament formation by profilinactin complexes to their proline-rich binding partners (28). For example, EVH1-binding motifs might facilitate interaction with Protein-enabled homolog (ENAH) and Vasodilator stimulated phosphoprotein (VASP) PCARE interactor proteins that we have identified in yeast two-hybrid screening and tandem affinity purification studies (9). A very recent structural study has shown that PCARE binds with a high affinity to the EVH1 domain of the protein ENAH, which as noted we had previously identified as a PCARE interactor, that plays a role in actin polymerization and cytoskeletal remodelling (29). Taken together, these studies establish that PCARE acts through ENAH to regulate actin filament assembly in the eye photoreceptor cilium.
MAK/RP62 kinase candidate phosphorylation site mutants PCARE RP62a and PCARE RP62b showed a similar frequency of expansion formation to the wildtype. Possibly, that although MAK/RP62 kinase is crucial for ciliary length and retinal health, it does not directly affect actin network polymerization via PCARE. However, for PCARE RP62a and PCARE RP62b, we observed a subtle but significant difference between hTERT RPE-1 and IMCD-3 cell lines in the ciliary tip expansion size.
When comparing the results obtained in IMCD-3 and hTERT RPE-1 cells, it is clear that although most of the findings were evident in both cell lines, the immortalized cell line models might provide contradictive readouts. Being derived from mouse kidney or human retinal epithelial cells, these models cannot fully represent the function of photoreceptor cells. Therefore, further confirmation of PCARE protein's functions is required with model systems that more faithfully represent the human retina, for example in human iPSC-derived retinal organoids (30). The recent advances in these techniques allow for precise editing of the gene of interest to induce mutations via the CRISPR-Cas9 editing machinery, which can be further studied at a different level of complexity such as protein expression, morphology or even function (20).
To conclude, all PCARE mutants that localized into the cilia formed ciliary tip expansions, suggesting that PCARE contains a redundancy of amino acid motifs via which it can interact with actin modulators and that it may interact with multiple actin-modulators. Our data also pinpoint the prominent role of proteins with EVH1 domains and hint towards the importance of palmitoylation lipid modification in the PCARE-actin interactions. This work presents a first glance at the importance of amino acid modules in PCARE and expands upon their structure and function in relationship to actin network modification, paving the way for improved understanding of mechanisms that underlie PCARE-associated retinal disease, as well as the formation of the OS disks of photoreceptors.

Bioinformatic prediction of structural and functional amino acid motifs within PCARE protein
The human PCARE SEQUENCE (UniProt:A6NGG8) was submitted to the Eukaryotic Linear Motif resource (http://elm.eu.org) server (13). Among the candidate matches were three proline-rich EVH1-binding SLiMs (805-809, 830-834, 1056-1060: http://elm.eu.org/elms/ LIG_EVH1_1.html). A multiple sequence alignment of vertebrate PCARE sequences was prepared and visualized with Jalview (31). All vertebrate PCARE sequences had multiple EVH1 motifs, with some variation in number and positioning, as often occurs with SLiMs. Since these candidates are in regions of predicted native disorder, amino acid conservation is driven by functional, not structural selection: Thus, these motifs are strong candidates for actin-regulatory function via EVH1 domain binding (32). By visual inspection, we also noted two conserved RPx[ST] motifs (513-516, 577-580) that are candidate phosphorylation sites (33) for the MAK (RP62) ciliary protein kinase (UniProt:P20794) required for long-term photoreceptor survival (33). All vertebrate PCARE sequences begin with the residues MetGlyCys indicative of N-terminal myristoylation and prenylation. Structural context was provided by native disorder predictors IUPRED (34) and DISOPRED (14) and the PSIPRED 4.0 (http://bioinf.cs.ucl.ac.uk/psipred/) workbench (16). More recently, the structural context was re-evaluated with AlphaFold (35), supporting the disorder predictions and providing high confidence assignment for the long helices (171 and 338) as an anti-parallel coiled coil pair (see Supplementary Material, Fig. S1).

Generation of mutant PCARE constructs
Human C2orf71/PCARE [NCBI GeneID:388939] full-length cDNA was obtained by PCR using Human Retina Marathon ® -Ready cDNA (Clontech) and cloned into Gateway™ pDONR201 vector (Thermo Fisher) as previously described (9). PCARE mutant constructs lacking functional and structural motifs were constructed by performing site-directed mutagenesis on pDONR with cloned PCARE cDNA construct with primer pairs that introduced deletions of nucleotide coding for the amino acid motifs via PCR amplification with Q5 High-fidelity DNA polymerase (New England Biolabs, M0491L). PCR reactions were performed using 0.2 ml PCR tubes in a final volume of 50 μl including the following components: 50 ng of template DNA, 0.5 μM of each primer (forward and reverse), 200 μM of each dNTPs, 1× Q5 Buffer and 1.6 U of Q5 High-fidelity DNA polymerase. 30 cycles of amplification were carried out under the following conditions: initial denaturation at 98 • C for 4 min, denaturation at 98 • C for 30 s, annealing at 50 • C for 30 s and extension at 72 • C for 3 min, following by final extension at 72 • C for 7 min. Primer sequences are listed in Supplementary Material, Table S1. Parental plasmid DNA was eliminated by DpnI treatment (1 μl of 20 U/μl) (New England Biolabs, R0176S) of a 10-μl aliquot of the linear amplification reaction for 1 at 37 • C. DNA was then directly transformed to competent Escherichia coli DH5α cells and purified with NucleoSpin Plasmid EasyPure (BIOKÉ, MN740727). Constructs were checked for deletions by amplification of the 200 nucleotide target region around the deletion with forward and reverse primers with Taq polymerase (Roche, 11 647 687 001). Thirty-five cycles of amplification were carried out under the following conditions: initial denaturation at 94 • C for 5 min, denaturation at 94 • C for 30 s, annealing at 58 • C for 30 s and extension at 72 • C for 20 s, following by final extension at 72 • C for 5 min. At the completion of the PCR, fragments were separated on 2.5% agarose gels containing 0.5 μg/ml ethidium bromide and further verified by Sanger sequencing. The entire coding region of constructs containing the modification of interest was verified by Sanger sequencing. The obtained entry clones were used to generate expression clones containing 3×HA tag using Gateway Technology (Life Technologies) and according to the manufacturer's instructions.

Statistical analysis
Statistical analyses were performed in Excel and Graphpad/Prism 6. An ALPACA tool (version 1.53c) was used for quantification of sizes of the ciliary tip expansions (38). Error bars represent standard deviations. An unpaired t test with Welch's correction was used to compare each mutant cilium tip expansions' sizes to the wild-type. N values are stated in the figures and represent cilia. Pvalues are stated in the figures and symbols indicate the following P-values: ns, P > 0.05; * P ≤ 0.05; * * P ≤ 0.01; * * * P ≤ 0.001.

Supplementary Material
Supplementary Material is available at HMG online.