A Conserved, Serine-Rich Protein Plays Opposite Roles in N-Mediated Immunity against TMV and N-Triggered Cell Death

Abstract

Plant nucleotide-binding, leucine-rich, repeat-containing proteins (NLRs) play important roles in plant immunity. NLR expression and function are tightly regulated by multiple mechanisms. In this study, a conserved serine/arginine-rich protein (SR protein) was identified through the yeast one-hybrid screening of a tobacco cDNA library using DNA fragments from the N gene, an NLR that confers immunity to tobacco mosaic virus (TMV). This SR protein showed an interaction with a 3' genomic regulatory sequence (GRS) and has a potential role in regulating the alternative splicing of N. Thus, it was named SR regulator for N, abbreviated SR4N. Further study showed that SR4N plays a positive role in N-mediated cell death but a negative role in N protein accumulation. SR4N also promotes multiple virus replications in co-expression experiments, and this enhancement may not function through RNA silencing suppression, as it did not enhance 35S-GFP expression in co-infiltration experiments. Bioinformatic and molecular studies revealed that SR4N belongs to the SR2Z subtype of the SR protein family, which was conserved in both dicots and monocots, and its roles in repressing viral immunity and triggering cell death were also conserved. Our study revealed new roles for SR2Z family proteins in plant immunity against viruses.

Keywords: N gene; RNA silencing; SR proteins; cell death; plant immunity; tobacco mosaic virus.

Figure 1

Interaction between NtaSR4N and the N gene in a yeast one-hybrid assay. (A–C) Diagrams of N promoter, Intron 3, and terminator DNA fragments used in bait vectors. (D) Point-to-point Y1H tests. Bait and prey vectors in each yeast strain are indicated to the left. Medium composition is indicated at the bottom.

Figure 2

Expression pattern of NtaSR4N. (A) Quantitative RT-PCR analysis of the NtaSR4N level during TMV infection in N plants. The Y-axis is an arbitrary unit of relative expression levels normalized to NtaTubulin. The X-axis is the number of hours after inoculation. (B) Detection of NbeSR4N expression via semiquantitative RT-PCR in transient assays. Combinations of vectors co-infiltrated are indicated on top of each lane. (C) Quantitation of DNA bands in (B) using ImageJ. A two-tailed Student’s t-test was used for comparison between the two samples. GraphPad Prism 8 was used for statistical analyses. (D) Expression levels of NtaSR4Na and b in 3- and 6-week-old plant leaves. Y-axis is the number of mRNA-seq reads per kb per million total reads (RPKM). Expression data represent mean values for three biological replicates (n = 3).

Figure 3

SR4N positively regulates cell death. (A) Cell death phenotype triggered by co-infiltration of N and p50 on N. benthamiana plants treated by control (left) and SR4N (right) VIGS vectors. Red circles mark the patches co-infiltrated with N and p50 Agrobacteria. (B) Statistical analysis of the cell death rate in the plants treated by control (left) and SR4N (right) VIGS vectors. Patches with HR/total infected patches indicate the cell death rate. A two-tailed Student’s t-test was used for comparison between the two samples using GraphPad Prism 8. (C) RT-PCR analysis of SR4N expression levels in VIGS experiments. Target genes are indicated to the left, and samples are indicated at the bottom of each lane. WT: wild-type untreated sample. (D) Cell death phenotype on SR4N overexpressed leaves. Infiltrated patches are marked by red circles, and vectors used for infiltration are indicated above each circle.

Figure 4

NtaSR4N promotes TMV replication but reduces N protein accumulation. (A) Green florescence imaging of agrobacteria-infiltrated N. benthamiana leaves under UV light. White circles mark the infiltrated patches with co-expressed genes indicated next to each circle. (B) Structure of N gene in pN2tag construct. Green arrow represents the native N promoter. Gray boxes marked I–III and IV–V represent fused exons I–III and exons IV–V of the wild-type N gene. The dashed box marks the modified alternative exon and 3′ ORF: AE18 and AE52 represent the 5′ 18- and 3′ 52-bp sequences of the 70-bp AE of wild = type N; TE168 represents a 168-bp SINE element from the tobacco genome (Supplementary Data S1); 3×FLAG, 9×Myc, and 6×His represent three-times Flag, nine-times Myc, and six = times His epitope tags, respectively. The open box represents the 3′ GRS of N. (C) Western blot and RT-PCR analysis of products from N and other genes in co-infiltration experiments. Constructs used in each sample are indicated on top of each lane. Target proteins are indicated to the left of the Western blots (top four panels). Target genes for RT-PCR analysis are indicated to the left of each gel image (bottom two panels). The two red * mark the position of N_L and N_S transcripts, respectively. (D) Quantification of N^tr/N ratio at the protein level (left) and N_L/N_S ratio at the transcript level (right). Blue columns represent data from NtaSR4Na-infiltrated samples, and brown columns represent data from NtaSR4Nb-infiltrated samples.

Figure 5

SR4N is conserved in both monocots and dicots. (A) Phylogeny tree of SR protein family from rice (blue IDs), Arabidopsis (orange IDs), tomato (purple IDs), and tobacco (green IDs) genomes. (B) Expression heatmap of RS2Z subclade members. Published RNA-seq data were obtained and analyzed (see Section 2.6). Sample sources are indicated on top of the heatmap: R, root; L, leaf; F, flower; S, seed. Gene IDs are indicated to the right. (C) Protein sequence alignments for the RS2Z subclade proteins. Blue line marks the RRM motif; red lines mark the zinc finger motifs; green line marks the serine/arginine-rich region; dark blue line marks the serine/proline-rich region.

Figure 6

SR4N functions are conserved in homologs from different plant species. (A) Impact of tomato, Arabidopsis, and rice SR4N on N protein and transcript accumulation. Co-expressed constructs are indicated on top of each lane. The SR4Ns expressed from the left to the right are SlySR4N, AthSR4Na, AthSR4Nb, OsaSR4Na, and OsaSR4Nb. Top four rows are Western blots, with target proteins indicated to the right. Bottom two rows are RT-PCR gels, with target transcripts indicated to the right. Red stars * mark the N_L and N_S isoforms. (B) Green florescence imaging of leaves in co-infiltration experiments. Yellow circles mark the infiltrated patches. Circle 1 is co-infiltrated with TMV-GFP and N; circle 2, TMV-GFP and EV; circle 3, TMV-GFP, N, and SR4N, indicated at the bottom. (C) Cell death triggered by SR4Ns from different plant species. Red circles mark the infiltrated patches, with vectors indicated above.

Figure 7

SR4N promotes the replication of multiple viruses but not the expression of the transgene. Yellow dashed circles mark the infiltrated patches. The viruses indicated to the left of each row and EV or SR4N indicated on top of each leaf were co-infiltrated. Images were taken at timepoint HPI are indicated to the right of each row. TBSV-GFP agrobacterium was infiltrated at a final OD600 of 0.02, TMV-GFP at 0.5, TRV1 and TRV2-GFP at 0.01, pMS4 at 0.05, and EV and SR4N agrobacteria at 0.1.