ABSTRACT
Xylem patterning in the root is established through the creation of opposing gradients of miRNAs and their targets, enabled by the cell-to-cell spread of the former. The miRNAs involved in xylem patterning, miR165/6, move through plasmodesmata, but how their trafficking is regulated remains elusive. Here, we describe that the receptor-like kinases BAM1/2 are required for the intercellular movement of miR165/6 in the stele and hence proper xylem patterning in the root.
MAIN TEXT
Tissue patterning in plant organ development depends primarily on positional information, which must be communicated between cells. Different mobile molecules can mediate cell-to-cell communication, including phytohormones, transcription factors, or peptides. In the past decade, multiple works have uncovered the relevance of small non-coding RNAs (sRNA) as mobile signaling molecules capable of acting as morphogens in plant development, determining leaf polarity, root vascular patterning, embryo meristem formation, female gametogenesis, and maintenance of the shoot apical meristem, regulating the acquisition of cell fate in a dose-dependent fashion (reviewed in 1-3). Interestingly, it has been recently shown that the cell-to-cell movement of microRNAs (miRNAs) is directional 4, indicating that this process must be precisely regulated.
An elegant example of how sRNA can determine pattern formation is provided by the study of xylem patterning in the root. Xylem patterning is established by a robust regulatory pathway comprising bidirectional cell signaling mediated by miRNAs 165 and 166 (miR165/6) and the transcription factors SHORT ROOT (SHR) and SCARECROW (SCR) 5: xylem precursors differentiate into two types of xylem vessels: metaxylem cells, with pitted secondary cell walls, in the centre of the vascular cylinder, and protoxylem cells, distinguishable by their spiral walls, in a peripheral position (Figure 1A). SHR is produced in the steel, and moves from cell to cell to the endodermis, where it activates SCR and, together with the latter, MIR165a and MIR166b. The resulting miR165 and miR166 move into the stele to pattern the class III HOMEODOMEIN-LEUCIN ZIPPER (HD-ZIP III) mRNA domains, particularly that of PHABULOSA (PHB), restricting them to the centre of the stele, which results in correct xylem patterning with formation of both metaxylem and protoxylem 5,6 (Figure 1A). Although miR165/6 have been shown to move symplastically through plasmodesmata7, how their trafficking is regulated remains elusive.
The plasma membrane- and plasmodesmata-localized receptor-like kinases BARELY ANY MERISTEM (BAM) 1 and 2 have been recently described as required for the cell-to-cell spread of RNA interference (RNAi) in the reporter SUC-SUL plants 8, in which production of mobile siRNA against the endogenous SULPHUR (SUL) gene in phloem companion cells causes non-cell autonomous silencing observable as a chlorotic phenotype around the leaf veins 9. Whether BAM1/2 also play a role in the cell-to-cell movement of other sRNAs, such as miRNAs, is yet to be determined.
In Arabidopsis roots, BAM1 is strongly and specifically expressed in the stele (Figure S1). We hypothesized that, considering this particular expression pattern, if BAM1 regulates movement of miRNA, it could mediate the cell-to-cell spread of miR165/6, hence acting as a regulator of xylem patterning. In order to determine whether BAM1/2 are required for correct xylem formation in the root, we observed xylem patterning in bam1/2 mutants 9,10. Interestingly, bam1 bam2 double mutants, but not bam1 or bam2 single mutants, display shorter roots (Figure S2) and show xylem defects consistent with a malfunction of miR165/6, namely absence of protoxylem files and overproliferation of metaxylem at the expense of protoxylem (Figure 1B and C; Figure S3). At the molecular level, bam1 bam2 mutants display increased levels of HD-ZIP III transcripts, but are not affected in the expression of MIR165/166, SHR, or SCR, or in the accumulation of miR165/6 (Figure 1D-F; Figure S4). Further supporting the idea that movement of miR165/6 is affected in the double mutants, the distribution of the PHB transcript is less restricted in the stele in the absence of BAM1/2 (Figure 1G), while lower levels of miR166 can be detected in this area (Figure 1H). On the contrary, transgenic plants overexpressing BAM1 have normal xylem and wild type-like accumulation of the HD-ZIP III transcripts (Figure S5). Taken together, these results indicate that BAM1/2 are required for proper xylem patterning, likely due to a function as positive regulators of the cell-to-cell movement of miR165/6.
The C4 protein from the geminivirus Tomato yellow leaf curl virus (TYLCV) interacts with the intracellular domain of BAM1/2 at the plasma membrane and has a negative impact on the cell-to-cell spread of RNAi 9. In order to see whether the activity of C4 can have an effect of xylem patterning, we observed the xylem in roots of transgenic plants expressing C4 under the control of the constitutive 35S promoter 9. Strikingly, expression of C4 led to xylem defects similar to those observed in bam1 bam2 mutants (Figure 2A and B). Plasma membrane localization of C4 is essential for this phenotype, since plants expressing the mutated version C4G2A, which loses its membrane association and localizes to chloroplasts exclusively 9, have wild type-like xylem (Figure S6). Transgenic plants expressing C4, but not C4G2A, have increased levels of HD-ZIP III transcripts (Figure 2C, Figure S6). However, C4 does not affect the expression of MIR166, SHR, or SCR, or the accumulation of miR165/6 (Figure 2D and E; Figure S7). As observed for bam1 bam2 mutants, the distribution of the PHB transcript in the stele is broader in the C4-expressing plants (Figure 2F), and lower levels of miR166 are detected in this part of the root (Figure 2F).
Since miR165/6 are produced in the endodermis, and from here traffic inwards into the stele establishing a gradient that determines HD-ZIP III dosage 5, we reasoned that if C4 is exerting its effect on xylem patterning through the interference with the cell-to-cell movement of miRNAs, then expressing C4 in the endodermis layer should have a non-cell autonomous effect and be sufficient to cause the observed phenotype. Indeed, transgenic plants expressing C4 under the control of the endodermis-specific SCR promoter display xylem patterning and related molecular phenotypes similar to those previously described for bam1 bam2 and 35S:C4 transgenic lines (Figure 2A-D; Figure S7), including a wider PHB domain and lower miR166 in the root stele (Figure 2F, G). Moreover, despite its localization in plasmodesmata 9, C4 does not disturb the movement of SHR-GFP (Figure S8). Therefore, C4 interferes with xylem patterning non-cell-autonomously, most likely through an impairment of miR165/6 movement. Of note, transgenic plants expressing C4 under the SCR promoter display wild type-like rosettes, but abnormal floral stems (Figure S9).
Recently, BAM1 was shown to act as a receptor for the CLE9/10 peptides to regulate periclinal cell division of xylem precursor cells 11. The results presented here unveil an additional, novel, redundant role of BAM1 and BAM2 in the regulation of xylem cell fate in the root stele. bam1 bam2 double mutants display defects in xylem patterning, which are mimicked cell-autonomously and non-cell-autonomously by the expression of the viral BAM1/2-interactor C4; however, all regulatory steps occurring upstream of the cell-to-cell movement of miR165/6 are unaltered in the absence of BAM1/2 or in the presence of C4. Despite normal accumulation of miR165/6, the action of these miRNAs on their target PHB is compromised in bam1 bam2 mutant or C4 transgenic lines, which correlates with a reduced distribution of miR166 in the root stele, underpinning the observed defective xylem patterning. Therefore, BAM1 and BAM2 seem to promote the cell-to-cell movement of both siRNA 9 and miRNA, an activity targeted by the viral effector C4; whether their role in sRNA-mediated intercellular communication underlies other biological functions of BAM1/2 remains to be determined.
Although our results provide novel insight into the mechanisms enabling the cell-to-cell movement of sRNA, which virtually impacts every aspect of plant biology, our current view of this process is still extremely limited and multiple questions remain to be answered. For example, whether sRNA travel in a free form or associated to proteins, or how directionality of the movement, if required, is accomplished, are long-standing questions. The elucidation of how BAM1/2 exert their role on the intercellular spread of sRNA at the molecular and cellular levels may shed light on these and other still elusive matters. However, it must be kept in mind that BAM1/2 are likely not the only proteins mediating the cell-to-cell movement of sRNA in plants: considering the restricted expression pattern of BAM1/2, together with the limited developmental phenotypes of the bam1 bam2 double mutants, additional molecular mechanisms must regulate this process outside the BAM1/2 expression domains.
METHODS
Plant materials and growth conditions
Mutants and transgenic plants used in this study are summarized in Table S1. Seedlings used for quantitative RT-PCR (qRT-PCR) and xylem phenotype analysis were grown on half strength Murashige and Skoog (1/2MS) medium containing 1% sucrose and 1% agar. Plates were placed vertically in a growth chamber with a photoperiod of 16 h light/8 h dark at 22°C. SCR:C4 plants used for phenotyping were grown in soil under the same environmental conditions described above.
Real-time quantitative RT-PCR (qRT-PCR)
For real-time quantitative RT-PCR (qRT-PCR), total RNA was extracted using Plant RNA Kit (Omega, USA) and reverse-transcribed by First Chain cDNA Synthesis Kit (TonkBio, China). qPCR was performed using C1000 Touch Thermal Cycler (Bio-Rad, USA); 20μl of PCR reaction mixture contained 10μl of SYBR Green mix (Bio-Rad, USA), 1μl of primer mix (10μM), 1μl reverse-transcribed product and 8μl of water. ACTIN (ACT2) was used as normalizer. Data were analyzed using the 2ΔΔCT method. To quantify the accumulation of miR166, stem-loop qPCR was conducted as previously described12. All primers used for qPCR are listed in Table S2.
Constructs and generation of transgenic lines
To generate the pSCR:C4 construct, the coding sequence of C4 was cloned into pENTR/D-TOPO (Invitrogen, USA), and subsequently Gateway-cloned into the pSCR:GW vector13 through an LR reaction (Invitrogen, USA). A. thaliana plants were transformed using the floral dipping method14.
In situ hybridization
In situ hybridization was performed as previously described15,16. The probe for PHB detection was cloned into the pGEM-T Easy vector (Promega, USA), using the primers listed in Table S2. For microRNA in situ hybridization, a specific miR166 LNA probe (QIAGEN, Germany) was used. 100 ng probe were used per slide. The hybridization temperature was 52°C for PHB detection, and 58°C for miR166 detection.
Small RNA (sRNA) sequencing
Small-RNA (sRNA) data analyses were performed using a pipeline previously described17. Briefly, raw reads were trimmed using trim_galore v0.4.0 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to remove the adapter sequences and bases that have a quality score lower than 10. Reads that could not be aligned to structural RNA sequences (rRNA, tRNA, snoRNA, snRNA, etc.) were aligned to the TAIR10 genome using Burrows–Wheeler aligner by allowing one mismatch per read17. The Tair10 genome was divided into non-overlapping 200-bp bins. The number of sRNA reads (with different lengths) in each 200-bp bin or specific genes were summarized and normalized to the structural RNA-removed library size (reads per 10 million) using bedtools v2.26.0 (https://bedtools.readthedocs.io/en/latest/). Results from two independent transgenic lines per construct were pooled.
Confocal imaging
All confocal images were acquired using a Leica TCS SP8 point scanning confocal microscope. For basic fuchsin staining, 5- or 6-day-old seedlings were first treated with 1M KOH solution for 6 hours at 37°C. Seedlings were then stained with 0.01% basic fuchsin solution in water for 5 minutes, and subsequently destained in 70% ethanol for 10 minutes. To check BAM1 expression pattern and SHR-GFP movement in the root tip, 5-day-old seedlings were imaged after propidium iodide (PI) staining. The settings used for the laser scanning are as follows: Ex:561nm, Em:600-700nm for basic fuchsin staining; Ex:488nm, Em:500-550nm for GFP; Ex:514nm, Em:525-570nm for YFP; Ex:561nm, Em:-680 nm for PI staining.
ACKNOWLEDGEMENTS
The authors thank Steven Clark and Zachary Nimchuk for kindly sharing materials; Wenjie Zeng, Xinyu Jian, Aurora Luque, and Yujing (Ada) Liu for technical assistance; and all members in Rosa Lozano-Duran’s and Alberto Macho’s groups for stimulating discussions and helpful suggestions. This research was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences, Grant No. XDB27040206, and by the National Natural Science Foundation of China (NSFC) (grant numbers 31671994 and 31870250). Research in RL-D’s lab is funded by the Shanghai Center for Plant Stress Biology of the Chinese Academy of Sciences and the 100 Talent program of the Chinese Academy of Sciences. We apologize to authors of relevant primary research works that could not be directly cited in this manuscript due to length restrictions.