Transfer of endogenous small RNAs between branches of scions and rootstocks in grafted sweet cherry trees

Abstract

Grafting is a well-established agricultural practice in cherry production for clonal propagation, altered plant vigor and architecture, increased tolerance to biotic and abiotic stresses, precocity, and higher yield. Mobile molecules, such as water, hormones, nutrients, DNAs, RNAs, and proteins play essential roles in rootstock-scion interactions. Small RNAs (sRNAs) are 19 to 30-nucleotides (nt) RNA molecules that are a group of mobile signals in plants. Rootstock-to-scion transfer of transgene-derived small interfering RNAs enabled virus resistance in nontransgenic sweet cherry scion. To determine whether there was long-distance scion-to-rootstock transfer of endogenous sRNAs, we compared sRNAs profiles in bud tissues of an ungrafted 'Gisela 6' rootstock, two sweet cherry 'Emperor Francis' scions as well as their 'Gisela 6' rootstocks. Over two million sRNAs were detected in each sweet cherry scion, where 21-nt sRNA (56.1% and 55.8%) being the most abundant, followed by 24-nt sRNAs (13.1% and 12.5%). Furthermore, we identified over three thousand sRNAs that were potentially transferred from the sweet cherry scions to their corresponding rootstocks. In contrast to the sRNAs in scions, among the transferred sRNAs in rootstocks, the most abundant were 24-nt sRNAs (46.3% and 34.8%) followed by 21-nt sRNAs (14.6% and 19.3%). In other words, 21-nt sRNAs had the least transferred proportion out of the total sRNAs in sources (scions) while 24-nt had the largest proportion. The transferred sRNAs were from 574 cherry transcripts, of which 350 had a match from the Arabidopsis thaliana standard protein set. The finding that "DNA or RNA binding activity" was enriched in the transcripts producing transferred sRNAs indicated that they may affect the biological processes of the rootstocks at different regulatory levels. Overall, the profiles of the transported sRNAs and their annotations revealed in this study facilitate a better understanding of the role of the long-distance transported sRNAs in sweet cherry rootstock-scion interactions as well as in branch-to-branch interactions in a tree.

Fig 1

Experimental design and sample collection (A) and small RNA profiles in sweet cherry scions (B).

Fig 2

Summary of sRNAs (A) and gene networks of overrepresented sRNAs in bud tissues of sweet cherry scion 15 (Scion15) ‘Emperor Francis’ grafted on a ‘Gisela 6’ rootstock (RS15). The ontology file of GO_FULL in BiNGO and A. thaliana annotation were used as the references to identify overexpressed GO terms (P < 0.05). Bubble color indicates the P-value. Overrepresented sRNAs in “Biological process” (B) and “Molecular function” (C). No overrepresented GO terms are present in “Cellular component”.

Fig 3. A diagram showing the pipeline of determining scion-to-rootstock transferred sRNAs.

Fig 4. Profiles of putative sRNAs transferred from sweet cherry scions to rootstocks.

RS15_specific and RS19_specific are sRNAs found in the grafted rootstocks, RS15 and RS19, but absent in the ungrafted.