Arabidopsis immune responses triggered by cellulose‐ and mixed‐linked glucan‐derived oligosaccharides require a group ofleucine‐rich repeat malectinreceptor kinases

[EN] The plant immune system perceives a diversity of carbohydrate ligands from plant and microbial cell walls through the extracellular ectodomains (ECDs) of pattern recognition receptors (PRRs), which activate pattern-triggered immunity (PTI). Among these ligands are oligosaccharides derived from mixed-linked b- 1,3/b-1,4-glucans (MLGs; e.g. b-1,4-D-(Glc)2-b-1,3-D-Glc, MLG43) and cellulose (e.g. b-1,4-D-(Glc)3, CEL3). The mechanisms behind carbohydrate perception in plants are poorly characterized except for fungal chitin oligosaccharides (e.g. b-1,4-D-(GlcNAc)6, CHI6), which involve several receptor kinase proteins (RKs) with LysM-ECDs. Here, we describe the isolation and characterization of Arabidopsis thaliana mutants impaired in glycan perception (igp) that are defective in PTI activation mediated by MLG43 and CEL3, but not by CHI6. igp1–igp4 are altered in three RKs – AT1G56145 (IGP1), AT1G56130 (IGP2/IGP3) and AT1G56140 (IGP4) – with leucine-rich-repeat (LRR) and malectin (MAL) domains in their ECDs. igp1 harbors point mutation E906K and igp2 and igp3 harbor point mutation G773E in their kinase domains, whereas igp4 is a T-DNA insertional loss-of-function mutant. Notably, isothermal titration calorimetry (ITC) assays with purified ECDRKs of IGP1 and IGP3 showed that IGP1 binds with high affinity to CEL3 (with dissociation constant KD = 1.19 0.03 lM) and cellopentaose (KD = 1.40 0.01 lM), but not to MLG43, supporting its function as a plant PRR for cellulose-derived oligosaccharides. Our data suggest that these LRR-MAL RKs are components of a recognition mechanism for both cellulose- and MLG-derived oligosaccharide perception and downstream PTI activation in Arabidopsis.SIGrant PID-2021-126006OB-100 from the Spanish Ministry of Science and Innovation to AMThis work has also been financially supported by the ‘Severo Ochoa (SO) Programme for Centres of Excellence in R&D’ from the Agencia Estatal de Investigaci on (AEI) of Spain (grants SEV-2016-0672 (2017-2021) and CEX2020-000999-S (2022-2025) to the CBGP). In the frame of the SO program, HM and PF-C were supported with postdoctoral fellowships. MM-D, DJB and DR were recipients of PhD Fellows PRE2019-088120 and PRE2019-091276 (SEV-2016- 0672) from AEI, and IND2017/BIO-7800 from Madrid Regional Government, respectively. Research in the lab of JS was financially supported by the University of Lausanne, the European Research Council (ERC) (grant agreement no. 716358) and the Swiss National Science Foundation (grant no. 310030_204526)

Differential Expression of Fungal Genes Determines the Lifestyle of Plectosphaerella Strains During Arabidopsis thaliana Colonization

16 Päg.The fungal genus Plectosphaerella comprises species and strains with different lifestyles on plants, such as P. cucumerina, which has served as model for the characterization of Arabidopsis thaliana basal and nonhost resistance to necrotrophic fungi. We have sequenced, annotated, and compared the genomes and transcriptomes of three Plectosphaerella strains with different lifestyles on A. thaliana, namely, PcBMM, a natural pathogen of wild-type plants (Col-0), Pc2127, a nonpathogenic strain on Col-0 but pathogenic on the immunocompromised cyp79B2 cyp79B3 mutant, and P0831, which was isolated from a natural population of A. thaliana and is shown here to be nonpathogenic and to grow epiphytically on Col-0 and cyp79B2 cyp79B3 plants. The genomes of these Plectosphaerella strains are very similar and do not differ in the number of genes with pathogenesis-related functions, with the exception of secreted carbohydrate-active enzymes (CAZymes), which are up to five times more abundant in the pathogenic strain PcBMM. Analysis of the fungal transcriptomes in inoculated Col-0 and cyp79B2 cyp79B3 plants at initial colonization stages confirm the key role of secreted CAZymes in the necrotrophic interaction, since PcBMM expresses more genes encoding secreted CAZymes than Pc2127 and P0831. We also show that P0831 epiphytic growth on A. thaliana involves the transcription of specific repertoires of fungal genes, which might be necessary for epiphytic growth adaptation. Overall, these results suggest that in-planta expression of specific sets of fungal genes at early stages of colonization determine the diverse lifestyles and pathogenicity of Plectosphaerella strains.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) grant BIO2015-64077-R and the Spanish Research Agency (AEI) grant RTI2018-096975-B-I00 to A. Molina and by the “Severo Ochoa Programme for Centers of Excellence in R&D” grant SEV-2016-0672 (2017-2021) to the CBGP (UPM-INIA). In the frame of SEV-2016-0672 program, H. Mélida was supported with a postdoctoral contract. A. Muñoz-Barrios was financially supported by the Universidad Politécnica de Madrid (UPM) Ph.D. students PIF program, I. del Hierro was a FPU fellow (Spanish Ministry of Education, Culture and Sports grant FPU16/07118), V. Fernández-Calleja was supported by the Consejería de Educacíon e Investigacíon of Comunidad de Madrid YEI program for postdoctoral researchers (PEJD-2016/BIO-3327), and the work was further supported through a Comunidad de Madrid YEI program for laboratory technicians grant (PEJ16/BIO/TL-1570).Peer reviewe

Arabidopsis immune responses triggered by cellulose- and mixed-linked glucan-derived oligosaccharides require a group of leucine-rich repeat malectin receptor kinases

18 Päg.The plant immune system perceives a diversity of carbohydrate ligands from plant and microbial cell walls through the extracellular ectodomains (ECDs) of pattern recognition receptors (PRRs), which activate pattern-triggered immunity (PTI). Among these ligands are oligosaccharides derived from mixed-linked β-1,3/β-1,4-glucans (MLGs; e.g. β-1,4-D-(Glc)2 -β-1,3-D-Glc, MLG43) and cellulose (e.g. β-1,4-D-(Glc)3 , CEL3). The mechanisms behind carbohydrate perception in plants are poorly characterized except for fungal chitin oligosaccharides (e.g. β-1,4-d-(GlcNAc)6 , CHI6), which involve several receptor kinase proteins (RKs) with LysM-ECDs. Here, we describe the isolation and characterization of Arabidopsis thaliana mutants impaired in glycan perception (igp) that are defective in PTI activation mediated by MLG43 and CEL3, but not by CHI6. igp1-igp4 are altered in three RKs - AT1G56145 (IGP1), AT1G56130 (IGP2/IGP3) and AT1G56140 (IGP4) - with leucine-rich-repeat (LRR) and malectin (MAL) domains in their ECDs. igp1 harbors point mutation E906K and igp2 and igp3 harbor point mutation G773E in their kinase domains, whereas igp4 is a T-DNA insertional loss-of-function mutant. Notably, isothermal titration calorimetry (ITC) assays with purified ECD-RKs of IGP1 and IGP3 showed that IGP1 binds with high affinity to CEL3 (with dissociation constant KD  = 1.19 ± 0.03 μm) and cellopentaose (KD  = 1.40 ± 0.01 μM), but not to MLG43, supporting its function as a plant PRR for cellulose-derived oligosaccharides. Our data suggest that these LRR-MAL RKs are components of a recognition mechanism for both cellulose- and MLG-derived oligosaccharide perception and downstream PTI activation in Arabidopsis.This work was supported by grant RTI2018-096975-B-I00 from the Spanish Ministry of Science, Innovation and Universities to AM and grant PID-2021-126006OB-100 from the Spanish Ministry of Science and Innovation to AM. This work has also been financially supported by the ‘Severo Ochoa (SO) Programme for Centres of Excellence in R&D’ from the Agencia Estatal de Investigación (AEI) of Spain (grants SEV-2016-0672 (2017-2021) and CEX2020-000999-S (2022-2025) to the CBGP). In the frame of the SO program, HM and PF-C were supported with postdoctoral fellowships. MM-D, DJB and DR were recipients of PhD Fellows PRE2019-088120 and PRE2019-091276 (SEV-2016-0672) from AEI, and IND2017/BIO-7800 from Madrid Regional Government, respectively. Research in the lab of JS was financially supported by the University of Lausanne, the European Research Council (ERC) (grant agreement no. 716358) and the Swiss National Science Foundation (grant no. 310030_204526).With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2020‐000999‐S)Peer reviewe

Trends in soil mercury stock associated with pollution sources on a Mediterranean island (Majorca, Spain)

8 Pág. Centro de Biotecnología y Genómica de PlantasHg is a global concern given its adverse effects on human health, food security and the environment, and it requiring actions to identify major local Hg sources and to evaluate pollution. Our study provides the first assessment of Hg stock trends on the entire Majorca surface, identifying major Hg sources by studying the spatiotemporal soil Hg variation at two successive times (2006 and 2016-17). The Hg soil concentration ranged from 14 to 258 μg kg-1 (mean 52 μg kg-1). Higher concentrations (over 100 μg kg-1) were found in two areas: (i) close to the Alcudia coal-fired power plant; (ii) in the city of La Palma. During the 11-year, the total Hg stock in Majorcan soil increased from 432.96 tons to 493.18 tones (14% increase). Based on a block kriging analysis, soil Hg enrichment due to power plant emissions was clearly detectable on a local scale (i.e. a shorter distance than 18 km from the power plant). Nonetheless, a significant island-wide Hg increase due to diffuse pollution was reported. This result could be extrapolated to other popular tourist destinations in the Mediterranean islands where tourism has increased in recent decades In short, more than 60 tons of Hg have accumulated on Majorca island in 11 years.We greatly appreciate the financial assistance provided by Spanish Ministry of Science and Innovation (Project CGL2013-43675-P) and CAM (Project AGRISOST-CM S2018/BAA-4330).Peer reviewe