Salicylic acid and RNA interference mediate antiviral immunity of plant stem cells

Significance Plant viruses, like those infecting animals, threaten the health of their hosts, can spread rapidly and globally, and challenge agricultural productivity in many species. Understanding antiviral defense and keeping plants virus-free is therefore of the utmost importance. Virus exclusion from stem cells is not only relevant for the infected individual and the potential to recover from acute infection but is believed to also block infection of the host germline, ultimately preventing vertical transmission of disease from parent to offspring and spread of viral infections via seeds. In this paper, we describe a stem cell–specific antiviral pathway in plants, which is of significant biological and economic relevance as it maintains stem cells virus-free.


Figure S1: (A)
Examples of main inflorescence shoot apical meristems (SAMs) from TuMV-6K2:Scarlet-infected WT and rdr1 mutant transgenic lines expressing nuclear fluorescent reporter H2B:Clover in the stem cell domain of the SAM, through the pCLV3 promoter, at 16-17 dpi.This color scheme is used for laser confocal microscopy images throughout this study: DAPI fluorescence is in grayscale, Clover is in green (except in Fig. 2D, E), Scarlet fluorescence is in orange-to-yellow (FIJI LUT "OrangeHot", with increasing signal intensity going from orange to yellow).Scale bars always indicate 20 m.The white arrowheads delineate the "core" virus exclusion zone in WT plants, the two top-most cell layers: L1 and L2.(B) As in (A), but WT plants after mock-inoculation (top) or infection with TuMV-6K2:Scarlet.Table S3: primers used in this study, for the purposes indicated on the left.Genotyping primers arefound in Table S2

Figure S2 :
Figure S2: Plots showing fluorescence values over time, every 2 days from 11 to 15 days post-inoculation (dpi), in the top 35 m of the SAM of (from left to right) WT, rdr6, rdr1 and rdr1rdr6 knock-out mutants (right).The green line indicates the average Clover fluorescence and identifies the L1-L2-L3 cell layers, the dark magenta line indicates the average Scarlet fluorescence (normalized in each sample to the respective Clover values), while the light magenta area indicates the standard deviation of Scarlet fluorescence among the samples.N indicates the number of shoot apical meristems analyzed.

Figure S3 :
Figure S3: (A) Photos of plants one month after inoculation with TuMV-6K2:Scarlet.Top: all infected plants lose apical dominance, but WT generate fertile shoots from axillary meristems, while rdr1 mutant plants do not.Bottom: rdr1 mutants complemented with WT (left) or catalytically inactive D801A (right) alleles of Clover-tagged RDR1.Two independent transgenic lines per construct are shown.(B) RT-qPCR analysis to quantify RDR1 mRNA in the RDR1 complementation lines indicated.The primers used span the T-DNA insertion in rdr1-1.Dots indicate technical replicates.

Figure S4 :
Figure S4: (A) PAGE northern blot detection of TuMV 6K2-derived sRNA in systemically infected WT and rdr1 plants, 11 dpi.Biological duplicates are shown, each sample being a pool of tissue from 4-5 plants.Mock-treated control (plants inoculated with buffer only) is on the left, snRNA U6 is used as loading control.(B) RT-qPCR analysis to quantify TuMV gRNA in the samples described in (A).Each column is a biological replicate, each dot a technical replicate.n.s.: p>0.05 (C) Bar plots showing the number of reads-per-million (divided into 21, 22 and 24 nt in length) of sRNA mapping to TuMV-6K2:Scarlet, as assessed by sRNA sequencing on duplicates of mock-and TuMV-infected apices (meristem and small flower buds) of WT and rdr1 plants.(D) Size distribution (18 to 26 nt length) and 5' nucleotide of the total reads in the experiment described in (C).

Figure S5 :
Figure S5: Plots showing two-fold increased (up) or decreased (down) accumulation of sRNA mapping to the Arabidopsis genome in mock-and TuMV-infected flower apices of WT and rdr1 plants, in pairwise comparisons indicated on the bottom horizontal axis.On the vertical axis the plots display the number of Arabidopsis genes from which the up-or down-regulated sRNA are derived.The plots have been divided according to sRNA size: 21nt (top), 22nt (middle) and 24nt (bottom).

Figure
Figure S6: (A)PAGE northern blot detection of Scarlet-derived sRNA in seedlings of WT and rdr1 transgenic lines expressing the siScar hairpin construct under the control of the pUBI promoter.Each sample is an independent transgenic line.snRNA U6 is used as loading control.(B) As in (A), but in flower apices (meristem and small flower buds) of rdr1 lines expressing siScar through the pCLV3 promoter (left) and seedlings expressing it through the pRDR1 promoter.Of note, the pCLV3-driven hairpin is processed only into 24 nt-long siRNA.

Figure S7 :
Figure S7: (A) Reads per million of small RNA of 21 or 22 nt derived from two methyltransferase genes indicated above, assessed by sRNA sequencing.For both sRNA clusters, WT TuMV vs. WT mock and rdr1 TuMV vs. WT TuMV pairwise comparisons revealed significant differences with adjusted p value < 0.05.(B) Transcripts per million (TPM) of WUS and the methyltransferase genes indicated above, assessed by smartseq3 mRNA sequencing on the same samples used for sRNA sequencing shown in Figures S4-S5 and above.None of the WT TuMV vs. WT mock and rdr1 TuMV vs. WT TuMV pairwise comparisons revealed differences with adjusted p value < 0.05 (n.s.).In (A) and (B), dots represent biological replicates.

Figure
Figure S8: (A) RT-qPCR quantification of P19 expression in upper stems and inflorences of the transgenic lines marked at the bottom, relative to pRDR1:P19 #3/1.Dots indicate technical replicates.(B) Laser confocal microscopy images of meristems from mock and TuMV-6K2:Scarlet-infected plants expressing H2B:Clover through the pUBI promoter.Expression is ubiquitous, including the L1 and L2 stem cell layers (white arrowheads).DAPI fluorescence in grayscale, Scarlet in orange-to-yellow.Scale bar: 20 m.

Figure
Figure S9: (A) Laser confocal microscopy images of meristems from WT, NahG, sid2 and dmr6 plants infected with TuMV-6K2:Scarlet.DAPI fluorescence in grayscale, Scarlet in orange-to-yellow.Scale bar: 20 m.(B) RT-qPCR quantification of PR1 expression in systemically infected tissues in the genotypes indicated after mock or TuMV-6K2:Scarlet inoculation.Each bar is a biological replicate of tissues from 4-5 plants, each dot is a technical replicate, relative abundance values are plotted on log10 scale.(C) RT-qPCR quantification of RDR1 expression in seedlings of WT, NahG and dmr6.Each bar is a biological replicate of 10+ seedlings, each dot is a technical replicate.(D) RT-qPCR quantification of PR1 expression in TuMV-6K2:Scarlet systemically infected tissues in the genotypes indicated.Each dot is a biological replicate of tissues from 4-5 plants, relative abundance values are plotted on log10 scale.(E) Quantification of RDR1 mRNA in the samples described in (D), with relative abundance values plotted on a linear scale.(B), (C), (D) and (E): n.s.: p>0.05; *: p<0.05; **: p<0.01, ***: p<0.001.(F) Laser confocal microscopy images of meristems from WT and npr1 plants infected with TuMV-6K2:Scarlet.DAPI fluorescence in grayscale, Scarlet in orange-to-yellow.Scale bar: 20 m.

Figure
Figure S11: (A) Photos of plants one month after inoculation with TuMV-6K2:Scarlet.The two rdr1 transgenic lines on the right express Clovertagged RDR1 through the pCLV3 promoter.WT and rdr1 controls are on the left.(B) As in (A), with two rdr1 transgenic lines expressing the siScar construct under the pCLV3 promoter (middle) or the pRDR1 promoter (right).(C) The lines described in (B), but infected with TuMV-6K2:Clover.(D) As in (A), of two transgenic lines expressing TBSV P19 through the pUBI promoter (left) and two under the pRDR1 promoter.
et al., PLOS ONE 2013 originally used to generate entry vectors for golden gate cloning, so contain additional adapters at 5' ends

Table S1 :
GreenGate entry vectors used to assemble the binary vectors in the left column.Previously available vectors are highlighted in yellow.

Table S2 :
Arabidopsis mutants used in this study (left) and the primers used for PCR genotyping (right)