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Additional file 1: Figure S1. Impact of loosenin-like proteins on NpXyn11A hydrolysis of azo-xylan in solution. Figure S2. Impact of loosenin-like proteins on cellulose/azo-xylan composites. Figure S3. In-solution hydrolysis of azo-xylan by Pm25 and its mutants. Figure S4. SDS-PAGE analysis of CmXyn10B (lane 2), NpXyn11A (lane 3) and TfXyn11A (lane 4). Figure S5. Replicate cellulose/azo-xylan composites after treatment for 1-h with (A) NpXyn11A or (B) TfXyn11A.

Additional file 3. Review history.

Additional file 1. Supplementary Figures (Haplotype Success and Potential Adaptation Fig. S1. Codon usage frequency of the SARS-CoV-2 coronavirus, the pangolin from which it likely evolved, and based on the alternative alleles from all currently known mutations. The TTT codon is significantly greater than expected based on pangolin (p < 0.01), suggesting that there is selection for this mutation.) (Structural analysis of SARS-CoV-2 mutants – nsp2 Fig. S2. Ab initio predicted structure of the N-terminal domain of nsp2. Sites of non-conservative substitution are depicted in yellow.) (Structural analysis of SARS-CoV-2 mutants – nsp3 Fig. S3. Predicted structure of the Arg/Lys-rich nucleic acid-binding domain of nsp3 (a.a. 1089-1201). Non-conservative substitutions are depicted in green. Positively charged residues are also shown, in yellow.) (Structural analysis of SARS-CoV-2 mutants – nsp13 Fig. S4. Structure of SARS-CoV-2 nsp13 based on PDB 6jyt. Non-conservative substitutions are depicted in green. Zinc ions are represented as yellow spheres.) (Structural analysis of SARS-CoV-2 mutants – Nucleocapsid Fig. S5. Sites of proteolytic cleavage of the Ser/Arg-rich motif of SARS-CoV2 nucleocapsid, predicted by the ExPASy PeptideCutter tool. The proteases, thermolysin (Therm), Arg-C proteinase (ArgC), proteinase K (ProtK), clostripain (Clost), and trypsin (Tryps), are assigned in their putative specific site of cleavage. The site of cleavage between Ser202 and Arg203 is marked with *.) (Structural analysis of SARS-CoV-2 mutants – Nucleocapsid Fig. S6. Crystal structure of SARS-CoV-2 nucleocapsid RNA-binding domain (PDB id 6vyo). Non-conservative substitutions are depicted in orange green. Zinc ions are represented as violet spheres.) (Structural analysis of SARS-CoV-2 mutants – Spike glycoprotein Fig. S7. Probability density of residues in the receptor-binding domain of SARS-CoV-2 forming contacts with the ACE2. The cutoff distance of 4 Å between any atom in a pair of residues was used. Bars with standard deviation higher than 50% of the probability density are considered transient interactions in the simulations and not included in the plot, except for the contact of residue 483, which is included for reference.) (Concluding remarks Fig. S8. Haplotype network produced with 9,294 sequences demonstrating the same relationships with the larger sample size presented in Fig. 2 (main text).) and Supplementary Tables (Table S1. The 107 sites with more than 2 alleles (more than one mutational event). The most frequent changes are provided first (column four) followed by second or third mutations, if present. The site in the genome is based on the reference sequence NC_0045512. Table S1 (cont.)) (Table S2. Groups of conservative substitutions considered in this study. Amino acids in brackets are evaluated after structural analysis.) (Table S3. Results from iRF-LOOP. Significant loci based on their ability to predict or be predicted by other loci. Three sites (21575_T, 11083_T, and 11074_T) appear to be hypermutation sites that occur as the result of long stretches of thymines due to consecutive runs of phenylalanine, leucine, and valine residues.) (Table S4. Summary of the main mutations, possible observable characteristics they may affect, and suggested future experiments to assess that.) (Supplementary References 1. Jia Z, Yan L, Ren Z, Wu L, Wang J, Guo J, et al. Delicate structural coordination of the Severe Acute Respiratory Syndrome coronavirus Nsp13 upon ATP hydrolysis. Nucleic Acids Res. 2019;47:6538–50. 2. von Brunn A, Teepe C, Simpson JC, Pepperkok R, Friedel CC, Zimmer R, et al. Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome. PLoS One. 2007;2:e459. 3. Neuman BW, Joseph JS, Saikatendu KS, Serrano P, Chatterjee A, Johnson MA, et al. Proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3. J Virol. 2008;82:5279–94. 4. Jia Z, Yan L, Ren Z, Wu L, Wang J, Guo J, et al. Delicate structural coordination of the Severe Acute Respiratory Syndrome coronavirus Nsp13 upon ATP hydrolysis. Nucleic Acids Res. 2019;47:6538–50. 5. He R, Dobie F, Ballantine M, Leeson A, Li Y, Bastien N, et al. Analysis of multimerization of the SARS coronavirus nucleocapsid protein. Biochem Biophys Res Commun. 2004;316:476–83.)

Ubiquitin proteasome system (UPS) is an ATP dependent pathway for targeted protein degradation. The role of UPS is to maintain a healthy protein balance in the cell and to mediate activation and repression of plant developmental processes, hormones and other signalling cascades as well as responses to environmental perturbations. The UPS is composed of several actors, the most important of them being the ubiquitin E3 ligases, which are responsible for providing the specificity for substrate recognition. About 5% of Arabidopsis thaliana genome encodes for ubiquitin E3 ligase genes (~1.400), classified in seven different subgroups, among which the second most abundant group is the RING-type ubiquitin E3 ligase family with nearly 500 members. More than half of the RING-type ubiquitin E3 ligase genes are uncharacterised. Those that are characterized demonstrate their multi-target ability which implies additional roles and cross-reactivity with other pathways. With the emergence of high throughput sequencing, improved Arabidopsis genome assemblies are available and there is a constantly growing amount of transcriptomics data available for the RING-type ubiquitin E3 ligase genes that link them to different developmental stages and perturbations. However, only few of these genes have been associated phenotypically with these processes. Our first aim was to use reverse genetics approach to rescreen Arabidopsis genome in order to update the number of annotated RING-type ubiquitin E3 ligase genes. We further aimed to develop a set of image-based phenotyping methods to systematically assign them in their signalling cascades and developmental pathways, and to functionally characterize the identified molecular networks of the RING-type ubiquitin E3 ligases and their substrates. This study revealed 50 new RING-type ubiquitin E3 ligases genes, while 31 earlier annotated genes were excluded, giving a total new count of 509 RING-type ubiquitin E3 ligases genes. RING-type ubiquitin E3 ligases were then assigned to different developmental, hormonal and/or perturbation related pathways, based on their gene expression profiles. To allow systematic and efficient functional confirmation of these associations in plants, protocols for image-based high throughput phenotypic assays were established. In these assays, the associated knockout lines were studied for rosette shape and growth, cotyledon emergence as a proxy for germination analysis, and Botrytis cinerea symptom progression. These phenotypic screens confirmed 36 flower enriched RING-type ubiquitin E3 ligases, 11 of which were associated to flowering, three rosette and one sepal development. Four of them responded negatively and one positively to ABA treatment at germination. Furthermore, ten RING-type ubiquitin E3 ligases were associated with Botrytis responses, with one characterised at molecular level. Collectively, the results of this study demonstrated the versatility and pleiotropy of RING-type ubiquitin E3 ligases and set the foundation for a systematic screening of phenotypes regulated by UPS components. RING-tyyppisen ubikitiin E3 -liigaasien monipuoliset roolit: käänteiset fenomiset lähestymistavat