The Natural Antisense Transcript DONE40 Derived from the lncRNA ENOD40 Locus Interacts with SET Domain Protein ASHR3 During Inception of Symbiosis in Arachis hypogaea.

The long noncoding RNA ENOD40 is required for cortical cell division during root nodule symbiosis (RNS) of legumes, though it is not essential for actinorhizal RNS. Our objective was to understand whether ENOD40 was required for aeschynomenoid nodule formation in Arachis hypogaea. AhENOD40 express from chromosome 5 (chr5) (AhENOD40-1) and chr15 (AhENOD40-2) during symbiosis, and RNA interference of these transcripts drastically affected nodulation, indicating the importance of ENOD40 in A. hypogaea. Furthermore, we demonstrated several distinct characteristics of ENOD40. (i) Natural antisense transcript (NAT) of ENOD40 was detected from the AhENOD40-1 locus (designated as NAT-AhDONE40). (ii) Both AhENOD40-1 and AhENOD40-2 had two exons, whereas NAT-AhDONE40 was monoexonic. Reverse-transcription quantitative PCR analysis indicated both sense and antisense transcripts to be present in both cytoplasm and nucleus, and their expression increased with the progress of symbiosis. (iii) RNA pull-down from whole cell extracts of infected roots at 4 days postinfection indicated NAT-AhDONE40 to interact with the SET (Su(var)3-9, enhancer of Zeste and Trithorax) domain containing absent small homeotic disc (ASH) family protein AhASHR3 and this interaction was further validated using RNA immunoprecipitation and electrophoretic mobility shift assay. (iv) Chromatin immunoprecipitation assays indicate deposition of ASHR3-specific histone marks H3K36me3 and H3K4me3 in both of the ENOD40 loci during the progress of symbiosis. ASHR3 is known for its role in optimizing cell proliferation and reprogramming. Because both ASHR3 and ENOD40 from legumes cluster away from those in actinorhizal plants and other nonlegumes in phylogenetic distance trees, we hypothesize that the interaction of DONE40 with ASHR3 could have evolved for adapting the nodule organogenesis program for legumes.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Segregation of nod-containing and nod-deficient bradyrhizobia as endosymbionts of Arachis hypogaea and as endophytes of Oryza sativa in intercropped fields of Bengal Basin, India.

Bradyrhizobial invasion in dalbergoid legumes like Arachis hypogaea and endophytic bacterial invasions in non-legumes like Oryza sativa occur through epidermal cracks. Here, we show that there is no overlap between the bradyrhizobial consortia that endosymbiotically and endophytically colonise these plants. To minimise contrast due to phylogeographic isolation, strains were collected from Arachis/Oryza intercropped fields and a total of 17 bradyrhizobia from Arachis (WBAH) and 13 from Oryza (WBOS) were investigated. 16SrRNA and concatenated dnaK-glnII-recA phylogeny clustered the nodABC-positive WBAH and nodABC-deficient WBOS strains in two distinct clades. The in-field segregation is reproducible under controlled conditions which limits the factors that influence their competitive exclusion. While WBAH renodulated Arachis successfully, WBOS nodulated in an inefficient manner. Thus, Arachis, like other Aeschynomene legumes support nod-independent symbiosis that was ineffectual in natural fields. In Oryza, WBOS recolonised endophytically and promoted its growth. WBAH however caused severe chlorosis that was completely overcome when coinfected with WBOS. This explains the exclusive recovery of WBOS in Oryza in natural fields and suggests Nod-factors to have a role in counterselection of WBAH. Finally, canonical soxY1 and thiosulphate oxidation could only be detected in WBOS indicating loss of metabolic traits in WBAH with adaptation of symbiotic lifestyle.