The essential chloroplast ribosomal protein uL15c interacts with the chloroplast RNA helicase ISE2 and affects intercellular trafficking through plasmodesmata

Abstract

Chloroplasts retain part of their ancestral genomes and the machinery for expression of those genomes. The nucleus-encoded chloroplast RNA helicase INCREASED SIZE EXCLUSION LIMIT2 (ISE2) is required for chloroplast ribosomal RNA processing and chloro-ribosome assembly. To further elucidate ISE2's role in chloroplast translation, two independent approaches were used to identify its potential protein partners. Both a yeast two-hybrid screen and a pull-down assay identified plastid ribosomal protein L15, uL15c (formerly RPL15), as interacting with ISE2. The interaction was confirmed in vivo by co-immunoprecipitation. Interestingly, we found that rpl15 null mutants do not complete embryogenesis, indicating that RPL15 is an essential gene for autotrophic growth of Arabidopsis thaliana. Arabidopsis and Nicotiana benthamiana plants with reduced expression of RPL15 developed chlorotic leaves, had reduced photosynthetic capacity and exhibited defective chloroplast development. Processing of chloroplast ribosomal RNAs and assembly of ribosomal subunits were disrupted by reduced expression of RPL15. Chloroplast translation was also decreased, reducing accumulation of chloroplast-encoded proteins, in such plants compared to wild-type plants. Notably, knockdown of RPL15 expression increased intercellular trafficking, a phenotype also observed in plants with reduced ISE2 expression. This finding provides further evidence for chloroplast function in modulating intercellular trafficking via plasmodesmata.

Plant Cell-Cell Transport via Plasmodesmata Is Regulated by Light and the Circadian Clock

“…To identify molecular pathways that regulate PD transport, we and others have conducted forward genetic screens. These screens to identify factors controlling transport through PD have repeatedly revealed that chloroplasts influence PD transport (Provencher et al, 2001;Benitez-Alfonso et al, 2009;Zambryski, 2010, 2012;Burch-Smith et al, 2011;Stonebloom et al, 2012;Brunkard et al, 2013;Carlotto et al, 2016;Bobik et al, 2019). This has led to a paradigm shift, focusing less on the role of structural changes directly at PD and more on how cellular physiology influences the function of PD.…”

A highly efficient protocol for transformingCuscuta reflexabased on artificially induced infection sites

“…In order to reveal whether the high transformation rates were a result of a specific susceptibility of C. reflexa to A. rhizogenes we exposed far-red light induced stems to Agrobacterium tumefaciens carrying a GFP gene in the T-DNA of a binary plasmid (Bobik et al, 2019) using the same setup. Only very weak background fluorescence was seen in this case in the orange channel and in the blue channel, while the high intensity of green fluorescence in a ring corresponding to the adhesive disk indicated that the GFP was expressed in this tissue as a result of the transformation (Fig.…”

“…As our experiments show, the protocol works with both, A. rhizogenes and A. tumefaciens and allows the expression of reporter proteins from different binary vectors and under the control of different promoters. The T-DNA of pRedRoot, a binary vector developed for A. rhizogenes , contains the dsRed protein under control of the Ubi10 promoter (Libiakova et al, 2018), while the A. tumefaciens line used by Bobik et al (2019) contains a GFP gene controlled by the 35S promoter. Both promoters allow strong constitutive expression of transgenes.…”

“…Cuscuta reflexa, Cuscuta campestris and Cuscuta platyloba were grown in a greenhouse on the host Pelargonium zonale under continuous illumination and a constant temperature of 21 °C (Förste et al, 2019). The bacteria and binary T-DNA-containing vectors pRedRoot and XM82, respectively, were kindly contributed by Prof. Harro Bouwmeester (University of Amsterdam, Netherlands) ( A. rhizogenes ) and Prof. Tessa Burch-Smith (University of Tennessee, USA) ( A. tumefaciens ) and are described in more detail in other studies (Limpens et al, 2004; Libiakova et al, 2018; Bobik et al, 2019). Cultures of A. rhizogenes MSU440 and A. tumefaciens GV3101 without binary plasmids were grown on LB medium (tryptone 10 g/L, NaCl 10 g/L, yeast extract 5 g/L, agar 7,5 g/L) supplemented with 100 mg/L Spectinomycin or 50 mg/L Rifampicin plus 50 mg/L Gentamycin, respectively.…”

The bRPS6-Family Protein RFC3 Prevents Interference by the Splicing Factor CFM3b during Plastid rRNA Biogenesis in Arabidopsis thaliana

“…prps21 is a viable mutant with a relatively strong defect in shoot growth and has reduced levels of 16S and 23S rRNAs [56]. Similarly, co-suppression lines of PRPL15 in Arabidopsis and a knockout line of prps15 in tobacco have shown reductions in 16S and 23S rRNA levels [57,58]. PALE CRESS (PAC) binds to 23S rRNA and promotes 50S subunit biogenesis, while the pac mutants dramatically reduce levels of both 16S and 23S rRNAs [59].…”