Transcriptome sequencing of rice leaves with blast symptoms collected from rice fields of the Philippines in 2017 and release of raw sequence data on OpenRiceBlast website for open access

<div>Infected rice samples were collected by Bo Zhou and team, and sent to the Sainsbury Laboratory for RNA extraction and sequencing. Library preparation and RNA-Seq sequencing runs were performed by Genewiz using Illumina HiSeq-2500 machines to produce paired-end reads with ~300 bp average insert size. Here we report the release of these data to general public with open access on OpenRiceBlast website.</div><div><br></div

Transcriptome sequencing of rice leaves with blast symptoms collected from rice fields of Bangladesh in 2017 and release of raw sequence data on OpenRiceBlast website for open access

<div>Infected rice samples were collected by Tofazzal Islam and team, and sent to the Sainsbury Laboratory for RNA extraction and sequencing. Library preparation and RNA-Seq sequencing runs were performed by Genewiz using Illumina HiSeq-2500 machines to produce paired-end reads with ~300 bp average insert size. Here we report the release of these data to general public with open access on OpenRiceBlast website.</div><div><br></div

The ZAR1 α1 helix can functionally replace the NRC2 and NRC3 α1 helix for Cf-4/Avr4-triggered hypersensitive cell death.

A) Structure of the ZAR1 resistosome with the N-terminal α1 helix highlighted. B) Schematic representation of ZAR1, NRC2, NRC3, and the respective NRC2ZAR1α1 and NRC3 ZAR1α1 chimaeras in which residues 1–17 and 1–21 are replaced by residues 1–17 from ZAR1, respectively. C) NRC ZAR1 α1 helix chimaeras accumulate to similar levels as wild-type NRC proteins. Immunoblot analysis of transient NRC-6xHA accumulation 5 days after agroinfiltration in wild-type N. benthamiana plants. D) NRC ZAR1 α1 helix chimaeras can complement Cf-4/Avr4 and Pto/AvrPto-triggered hypersensitive cell death in the N. benthamiana nrc2/3/4 CRISPR lines. Representative N. benthamiana leaves infiltrated with appropriate constructs were photographed 7–10 days after infiltration. The receptor/effector pair tested, Cf-4/Avr4 and Prf (Pto/AvrPto), are labelled above the leaf of NRC CRISPR line nrc2/3/4-210.4.3.1. The NRC tested, NRC2 and NRC3, are labelled on the leaf image. To ensure the NRC ZAR1 α1 helix chimaeras were not autoactive when expressed with either Cf-4 or Pto an EV control was taken along. A representative leaf of the independent nrc2/3/4-210.5.5.1 CRISPR line is shown in S8E Fig) Quantification of hypersensitive cell death. Cell death was scored based on a 0–7 scale between 7–10 days post infiltration. The results are presented as a dot plot, where the size of each dot is proportional to the count of the number of samples with the same score within each biological replicate. The experiment was independently repeated three times. The columns correspond to the different biological replicates. Significant differences between the conditions are indicated with an asterisk (*). Details of statistical analysis are presented in S8 Fig.</p

The N-terminal MADA motif of NRC3 is required for Cf-4/Avr4-triggered hypersensitive cell death.

A) NRC3 has an N-terminal MADA motif. Alignment of NRC2, NRC3, NRC4 and ZAR1 N-terminal MADA motif with the consensus sequence pattern of the MADA motif and the HMM score for each sequence. B) Schematic representation of NRC2, NRC3, and the respective NRC2L17E and NRC3L21E MADA mutants. C) NRC2 and NRC3 MADA mutants accumulate to similar levels as wild-type proteins. Immunoblot analysis of transiently expressed C-terminally 6xHA-tagged NRCs 5 days after agroinfiltration in wild-type N. benthamiana plants. D) NRC MADA mutants do not complement Cf-4/Avr4 or Pto/AvrPto-triggered hypersensitive cell death in the N. benthamiana nrc2/3/4 lines. Representative N. benthamiana leaves infiltrated with appropriate constructs were photographed 7–10 days after infiltration. The NRCs tested, NRC2 and NRC3, are labelled above the leaf of nrc2/3/4-210.4.3.1. The receptor/effector pair tested, Cf-4/Avr4 and Prf (Pto/AvrPto), are labelled on the leaf image. A representative leaf of the independent nrc2/3/4-210.5.5.1 line is shown in S7E Fig) Quantification of hypersensitive cell death. Cell death was scored based on a 0–7 scale between 7–10 days post infiltration. The results are presented as a dot plot, where the size of each dot is proportional to the count of the number of samples with the same score within each biological replicate. The experiment was independently repeated three times. The columns correspond to the different biological replicates. Significant differences between the conditions are indicated with an asterisk (*). Details of statistical analysis are presented in S7 Fig.</p

Statistical analysis of NRC ZAR1 α1 helix chimaera complementation of Cf-4/Avr4-triggered cell death <i>N</i>.

benthamiana nrc2/3/4 CRISPR lines. A) Statistical analysis was conducted using besthr R package [72]. The dots represent the ranked data and their corresponding means (dashed lines), with the size of each dot proportional to the number of observations for each specific value (count key below each panel). The panels on the right show the distribution of 100 bootstrap sample rank means, where the blue areas under the curve illustrate the 0.025 and 0.975 percentiles of the distribution. A difference is considered significant if the ranked mean for a given condition falls within or beyond the blue percentile of the mean distribution of the wild-type control. B) NRC ZAR1 α1 helix chimaeras can complement Cf-4/Avr4 and Pto/AvrPto-triggered hypersensitive cell death in the N. benthamiana nrc2/3/4 CRISPR lines. Representative N. benthamiana leaves infiltrated with appropriate constructs were photographed 7–10 days after infiltration. The receptor/effector pair tested, Cf-4/Avr4 and Prf (Pto/AvrPto), are labelled above the leaf of NRC CRISPR line nrc2/3/4-210.5.5.1. The NRCs tested, NRC2 and NRC3, are labelled on the leaf image. To ensure the NRC ZAR1 α1 helix chimaeras were not autoactive they were expressed with either Cf-4 or Pto and an EV control was taken along. (TIF)</p

In contrast to the widely conserved NRC3, NRC1 is not present in <i>N</i>. <i>benthamiana</i>.

Phylogenetic tree of the NRC-helper NLR family based on the full-length protein sequences was inferred using an approximately Maximum Likelihood method as implemented in FastTree [68] based on the Jones-Taylor-Thornton (JTT) model [71]. The tree was rooted on XP_00428175. The different NRC subfamilies are indicated. Domain architecture was determined using NLRtracker [3]. Probable pseudogenes or partial genes, including Nicotiana NRC1 are indicated. (TIF)</p

NRC3 complement Cf-2/Rcr3/Avr2-, Cf-5/Avr5-, and Cf-9/Avr9-triggered hypersensitive cell death in the <i>nrc2/3/4</i> knock-out lines.

A) Cf-2/Rcr3/Avr2-, Cf-5/Avr5-, and Cf-9/Avr9-triggered hypersensitive cell death is complemented in the nrc2/3/4 lines with NRC3, while Cf-9/Avr9-triggered hypersensitive cell death is partially complemented with NRC2. Representative N. benthamiana leaves infiltrated with appropriate constructs were photographed 7–10 days after agroinfiltration. The receptor/effector pair tested, Cf-2/Rcr3/Avr2, Cf-5/Avr5, and Cf-9/Avr9, are labelled above the leaf of nrc2/3/4-210.4.3.1. The NRC used for complementation or EV control are labelled on the leaf image, R3a/Avr3a was used as a positive control. B) Quantification of hypersensitive cell death. Cell death was scored based on a 0–7 scale between 7–10 days post infiltration. The results are presented as a dot plot, where the size of each dot is proportional to the count of the number of samples with the same score within each biological replicate. The experiment was independently repeated three times. The columns correspond to the different biological replicates. Significant differences between the conditions are indicated with an asterisk (*). C) Statistical analysis was conducted using besthr R package [72]. The dots represent the ranked data and their corresponding means (dashed lines), with the size of each dot proportional to the number of observations for each specific value (count key below each panel). The panels on the right show the distribution of 100 bootstrap sample rank means, where the blue areas under the curve illustrate the 0.025 and 0.975 percentiles of the distribution. A difference is considered significant if the ranked mean for a given condition falls within or beyond the blue percentile of the mean distribution of the wild-type control. (TIF)</p

Statistical analysis of Cf-4/Avr4-triggered cell death in different <i>N</i>.

benthamiana nrc mutant lines. A) Cf-4/Avr4-triggered cell death was scored on a scale of 0–7, with 0 being no response, and 7 being fully confluent cell-death in the entire infiltrated sector. Visible cell death starts appearing at a score of 4. B) Statistical analysis was conducted using the besthr R package. The dots represent the ranked data and their corresponding means (dashed lines), with the size of each dot proportional to the number of observations for each specific value (count key below each panel). The panels on the right show the distribution of 100 bootstrap sample rank means, where the blue areas under the curve illustrate the 0.025 and 0.975 percentiles of the distribution. A difference is considered significant if the ranked mean for a given condition falls within or beyond the blue percentile of the mean distribution of the wild-type control. C) Cf-4/Avr4-triggered hypersensitive cell death is lost in the nrc2/3 and nrc2/3/4 CRISPR lines. Representative N. benthamiana leaves infiltrated with appropriate constructs were photographed 7–10 days after infiltration. The NRC CRISPR lines, nrc2/3-209.3.3.1, nrc2/3/4-210.5.5.1, are labelled above the leaf and the receptor/effector pair tested, Cf-4/Avr4, Prf (Pto/AvrPto), Rpi-blb2/AVRblb2 or R3a/Avr3a, are labelled on the leaf image. Cf-4/EV and EV/Avr4 were also included. (TIF)</p

Hypersensitive cell death triggered by activation of cell-surface immune receptors requires the NRC network in Solanaceae.

Recognition of apoplastic effectors and translocated intracellular effectors by cell-surface leucine-rich repeat receptor proteins and intracellular NRC-type sensor-NLRs, respectively, results in NRC3-mediated hypersensitive cell death. This cell death requires an intact α1 helix of NRC3 indicating the possible formation of an activated NRC3 resistosome. Divergent pathogen effectors converge on this node to suppress both NLR triggered as well as cell-surface receptor triggered immune recognition.</p

Cf-4/Avr4-triggered hypersensitive cell death can be suppressed by divergent pathogen effectors.

A) A schematic representation of cell death suppression assay. A. tumefaciens containing vectors carrying both NRC3 and either SPRYSEC15, AVRcap1b, or mCherry as empty vector control, were co-expressed in N. benthamiana nrc2/3/4 CRISPR lines, with the effectors suppressing the cell death response. B) Cf-4/Avr4-triggered hypersensitive cell death is suppressed by SPRYSEC15 and AVRcap1b, but not the EV control. Representative image of N. benthamiana nrc2/3/4-210.4.3.1 CRISPR line leaves which were agroinfiltrated with NRC3/suppressor constructs, as indicated above the leaf, and either autoactive NRC3D480V, Prf (Pto/AvrPto), R3a/Avr3a, or Cf-4/Avr4 as labelled on the leaf image, photographed 7–10 days after infiltration. A representative leaf of the independent nrc2/3/4-210.5.5.1 CRISPR line is shown in S11C Fig) Quantification of hypersensitive cell death. Cell death was scored based on a 0–7 scale between 7–10 days post infiltration. The results are presented as a dot plot, where the size of each dot is proportional to the count of the number of samples with the same score within each biological replicate. The experiment was independently repeated three times. The columns correspond to the different biological replicates. Significant differences between the conditions are indicated with an asterisk (*). Details of statistical analysis are presented in S11 Fig.</p