IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)-like peptides and HAE (HAESA)-like receptors regulate corolla abscission in Nicotiana benthamiana flowers

Background Abscission is an active, organized, and highly coordinated cell separation process enabling the detachment of aerial organs through the modification of cell-to-cell adhesion and breakdown of cell walls at specific sites on the plant body known as abscission zones. In Arabidopsis thaliana, abscission of floral organs and cauline leaves is regulated by the interaction of the hormonal peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), a pair of redundant receptor-like protein kinases, HAESA (HAE) and HAESA-LIKE2 (HSL2), and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) co-receptors. However, the functionality of this abscission signaling module has not yet been demonstrated in other plant species. Results The expression of the pair of NbenIDA1 homeologs and the receptor NbenHAE.1 was supressed at the base of the corolla tube by the inoculation of two virus-induced gene silencing (VIGS) constructs in Nicotiana benthamiana. These gene suppression events arrested corolla abscission but did not produce any obvious effect on plant growth. VIGS plants retained a higher number of corollas attached to the flowers than control plants, an observation related to a greater corolla breakstrength. The arrest of corolla abscission was associated with the preservation of the parenchyma tissue at the base of the corolla tube that, in contrast, was virtually collapsed in normal corollas. In contrast, the inoculation of a viral vector construct that increased the expression of NbenIDA1A at the base of the corolla tube negatively affected the growth of the inoculated plants accelerating the timing of both corolla senescence and abscission. However, the heterologous ectopic overexpression of citrus CitIDA3 and Arabidopsis AtIDA in N. benthamiana did not alter the standard plant phenotype suggesting that the proteolytic processing machinery was unable to yield active peptides. Conclusion Here, we demonstrate that the pair of NbenIDA1 homeologs encoding small peptides of the IDA-like family and the receptor NbenHAE.1 control cellular breakdown at the base of the corolla tube awhere an adventitious AZ should be formed and, therefore, corolla abscission in N. benthamiana flowers. Altogether, our results provide the first evidence supporting the notion that the IDA-HAE/HSL2 signaling module is conserved in angiosperms. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-021-02994-8.

described in detail at the end of this Additional File.
Three cDNA libraries of N. benthamina corolla bases from flowers at developmental stage 4 collected from plants inoculated, respectively, with the empty clbv3' vector (CLBV) and the silencing constructs clbv3'-NibenIDA (IDAsil) and clbv3 '-NibenHAE (HAEsil) were prepared for Illumina Paired-End sequencing. After trimming and removing the adaptors, contamination and low-quality sequences, the Illumina sequencing generated 73,524,436 good reads that were mapped to the transcriptome and genome sequences of N. benthamiana (Bombarely et al. 2012). The Integrated Genome Viewer (IGV; Robinson et al., 2011;Thorvaldsdóttir et al., 2013) was used for visualization of short-read sequence alignments in single diagrams called Sashimi plots (Katz et al., 2015).
Since coding sequences (CDS) of NbenIDA1A and NbenIDA1B homeologs exhibited >92% nucleotide identity and the selected silencing trigger sequence of NbenIDA1B had >99% identity with that of NbenIDA1A (Additional File 2), it could be predicted that both homeologs might be silenced by clbv3'-NbenIDA VIGS construct. The BAM files for the three libraries mapped reads from the genomic regions of the NbenIDA1 pair of homeologs ( Figure 1). Reads of the CLBV and HAEsil libraries mapped the complete predicted sequence, without introns, of the NbenIDA1 pair of homeologs. However, reads in the IDAsil library only mapped a fragment of the sequence of both homeologs that matched the silencing trigger sequence of the clbv3'-NbenIDA construct. The coverage range for the IDAsil library was very high (>3000 and >1000, respectively, for each homeolog) suggesting that the silencing construct was very active in the corolla base at flower developmental stage 4. Therefore, the silencing construct clbv3'-NbenIDA appeared to be very efficient producing a strong knock-down effect on the expression of both NbenIDA1A and NbenIDA1B homeologs. The fragment of NbenHAE.1 selected as silencing trigger sequence showed >95% identity with that of NbenHAE.2 and contained stretches higher than 21 nucleotides with 100% identity at least in the first half of the sequence (Additional File 2). Therefore, it would be possible that both NbenHAE homeologs might be silenced. Reads of the CLBV and IDAEsil libraries mapped the complete predicted sequence of NbenHAE1 but those in the HAEsil library only mapped a fragment of the sequence that matched the silencing trigger sequence of the clbv3'-NbenHAE construct (Figure 2). The coverage range for the HAEsil library was very high (almost 27000) suggesting that as in the case of the other silencing construct, clbv3'-NbenHAE appeared to be very active for a strong knock-down effect on the expression of NbenHAE.1 in the corolla base at flower developmental stage 4. However, reads of all three libreries mapped the complete predicted sequence of NbenHAE.2 even though the silencing triggered sequence showed >95% identity (Additional File 2). Therefore, the silencing construct clbv3'-NbenHAE appeared to be very efficient producing only a strong knock-down effect on the expression of NbenHAE.1 but no effect on NbenHAE.2.

RNA-sequencing method
Total RNA from the base of corolla tubes in flowers from plants inoculated with the empty clbv3' vector and the silencing constructs clbv3'-NibenIDA and clbv3'-NibenHAE was isolated using acid phenol extraction and lithium chloride precipitation method as described in (Ecker and Davis, 1987). Quality of the isolated total RNA was checked and quantified using the NanoDrop (Thermo Fisher Scientific, Alcobendas, Madrid).
The isolated total RNA was used for library construction. Pair-end Libraries were prepared using the TruSeq™ RNA sample preparation kit (Illumina Inc.,) according to manufacturer's protocol. Briefly, 0.5 μg of total RNA was used for poly-A based mRNA enrichment selection using oligo-dT magnetic beads followed by fragmentation by divalent cations at elevated temperature resulting into fragments of 80-250 nt, with the major peak at 130 nt. First strand cDNA synthesis by random hexamers and reverse transcriptase was followed by the second strand cDNA synthesis performed using RNAseH and DNA Pol I. Double stranded cDNA was end repaired, 3'adenylated and the 3′-"T" nucleotide at the Illumina adaptor was used for the adaptor ligation. The ligation product was amplified with 15 cycles of PCR.
Each pair-end library was sequenced using TruSeq SBS Kit v3-HS, in paired end mode with the read length 2x76bp. A minimum of 50 million paired end reads for each sample were generated on HiSeq2000 (Illumina, Inc) following the manufacturer's protocol.
Images analysis, base calling and quality scoring of the run were processed using the manufacturer's software Real Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASSAVA. FASTQ files were pre-processed with Trimmomatic 0.38 (Bolger et al., 2014), and reads with average quality smaller than 25 and shorter than 36 bases were filtered.
The transcriptome and the genome sequences of N. benthamiana (Bombarely et al., 2012) were used as reference for sequence read mapping using the STAR RNA-seq aligner with default parameters (Dobien et al., 2013) as implemented in the OmicsBox suite (https://www.biobam.com/omicsbox). The mapping of short sequencing reads (FASTQ) was performed using RSEM software package, which allocates multi-mapping reads among transcripts using an expectation maximization approach (Li et al., 2011), based on a fast gapped-read alignment with Bowtie 2 (Langmead and Salzberg, 2012).