NB-LRR-encoding genes conferring susceptibility to organophosphate pesticides in sorghum

Organophosphate is the commonly used pesticide to control pest outbreak, such as those by aphids in many crops. Despite its wide use, however, necrotic lesion and/or cell death following the application of organophosphate pesticides has been reported to occur in several species. To understand this phenomenon, called organophosphate pesticide sensitivity (OPS) in sorghum, we conducted QTL analysis in a recombinant inbred line derived from the Japanese cultivar NOG, which exhibits OPS. Mapping OPS in this population identified a prominent QTL on chromosome 5, which corresponded to Organophosphate-Sensitive Reaction (OSR) reported previously in other mapping populations. The OSR locus included a cluster of three genes potentially encoding nucleotide-binding leucine-rich repeat (NB-LRR, NLR) proteins, among which NLR-C was considered to be responsible for OPS in a dominant fashion. NLR-C was functional in NOG, whereas the other resistant parent, BTx623, had a null mutation caused by the deletion of promoter sequences. Our finding of OSR as a dominant trait is important not only in understanding the diversified role of NB-LRR proteins in cereals but also in securing sorghum breeding free from OPS.

Crops growing under natural conditions face the threats from different environmental factors, among which pathogens and pests often cause a serious loss of yield [1][2][3] . To counteract such biotic stresses, breeding crops that confer resistance to them is demanding 4,5 . Plants are known to defense against pathogens by immune responses with R genes [6][7][8] . The majority of genetically characterized disease resistance traits map to R genes encoding nucleotide-binding domain (NB) and leucine-rich repeat (LRR) proteins, and they act as receptors to perceive effectors derived from pathogens and to activate effector-trigger immunity (ETI), which includes cell death and reactive oxygen species production [9][10][11][12][13][14][15][16] . In addition to harnessing these resistant genes, chemical management of pests is a practical way; plants are applied by the pesticides to manage the insects that attack a broad range of crop species 17,18 . Controlling aphids, thrips and other pests are important not only to avoid yield loss by severe infestation but also to prevent these insects from spreading vector-borne diseases [19][20][21][22] .
Organophosphates are a group of commonly used pesticides that kill insects by causing neurotoxicity through the inhibition acetylcholinesterase activity, whereas these compounds show no toxicity to host plants 23 . Cell death or leaf injury that resembles the hypersensitive reaction to pathogens, however, is rarely observed when crops are sprayed with a particular type of organophosphate pesticides 24 . Molecular analysis of such organophosphate pesticide sensitivity (OPS) was first reported in tomato; plants harboring the Pto locus (conferring resistance to Pseudomonas syringae pv. tomato strains) exhibited necrosis after being sprayed with fenthion, an organophosphate pesticide [25][26][27] . This OPS phenotype in tomato was shown to depend on the Fen gene, encoding a serinethreonine protein kinase belonging to the receptor-like kinases among the R genes 28 . More recently, another nucleotide-binding leucine-rich repeat (NB-LRR) gene termed Prf embedded within the Pto/Fen gene cluster was shown to act in concert with Pto/Fen to activate multiple plant-pathogen signal transduction pathways [29][30][31] .
The other example of NB-LRR responding to organic chemicals is VICTR in Arabidopsis, which responds to accession-specific root growth arrest with the small-molecule [5-(3,4-dichlorophenyl) furan-2-yl]-piperidine-1-ylmethanethione (DFPM) 32,33 . These findings present the intriguing recognition mechanism of external chemicals through NB-LRR, whereas the cases are limited to several species. In this study, we focused on another example of OPS in sorghum, which has segregated in a recombinant inbred population we recently established.

Results
Assessing organophosphate pesticide sensitivity. BTx623 and NOG displayed different responses to several types of organophosphate pesticides. Acephate (Ortran) was commonly used in our greenhouse to control aphids, and we noticed no visible growth defect throughout the entire growth period of both NOG and BTx623. In contrast, fenitrothion (Sumithion) and malathion (Marathion) caused severe necrosis only in NOG when applied to seedlings (Fig. 1a, chemicals structures of each pesticide are shown in Supplementary Fig. S1). This sensitivity was apparently holistic when fenitrothion was applied to field-growing NOG ( Supplementary  Fig. S2), excluding the possibility that OPS is confined developmentally. Necrotic lesions were also apparent Fine mapping. The BTx623xNOG RIL population consists of 213 individuals, in which the genotyping of 3710 markers was determined in the F 6 generation 39 . Displaying graphical genotypes of each RIL along with its OPS phenotype allowed us to narrow down the genomic region conferring OPS to a 743-kb region (Fig. 3a) between two SNP markers, Chr05:7,971,429 and Chr05:8,714,734. QTL analysis also showed that the QTL peaks were located at SNP marker Chr05: 8,666,663, encompassed by the chromosome regions detected from the recombination points (Fig. 3b). Within this region, there are 36 genes annotated in the database (https:// phyto zome. jgi. doe. gov, Phytozome version 12), in which a cluster of three NB-LRR genes (Sobic.005G071700, Sobic.005G071900, and Sobic.005G072000) existed in a tandem orientation (Fig. 3b, Supplementary Table S1). In fact, this cluster of three NB-LRR genes was reported as being likely to be responsible for leaf injury by Boyles et al. (2017) and for OPS of inbred line Nakei MS3B 46 . In this study, we termed it OSR in accordance with Kawahigashi et al. (2020) and further characterized these NB-LRR genes.

Characterization of the OSR locus in NOG.
We investigated the OSR locus in NOG, based on resequencing data of NOG we determined previously (DDBJ Sequence Read Archive Accession No. DRA008159). For simplicity, we termed three NB-LRR genes Sobic.005G071700, Sobic.005G071900, and Sobic.005G072000 as NLR-A, NLR-B, and NLR-C, respectively. First, comparison of these three genes with those in BTx623 showed that NLR-A was missing in NOG, lacking the corresponding genomic region from 8,457,439 to 8,557,269 on chromosome 5, which amounted to ~ 99.8 kb (Fig. 3b). Lack of NLR-A was subsequently confirmed by PCR specifically amplifying NLR-A (Fig. 3c). Given that OPS is dominant, involvement of NLR-A was unlikely (see below). Second, comparison of NLR-B and NLR-C indicated that both genes existed in NOG and BTx623. The Sorghum bicolor v3.1.1 database (https:// phyto zome. jgi. doe. gov, Phytozome version 12) predicted that NLR-B had no intron and encoded a 933 amino acid-long protein that had a nucleotide binding site domain (176-457 aa) and a leucine-rich repeat domain (483-903 aa). Similarly, NLR-C contained one exon of 2739 bp encoding 912 amino acids in both NOG and BTx623. We found that in NOG, NLR-B and NLR-C had two and ten amino-acid substitutions, respectively (Fig. 3d). For NLR-B, two amino-acid substitutions, I289V and S650R, were within the NB domain and LRR domain, respectively. The other polymorphism we detected in NOG was a 593-bp insertion, in the 5′ upstream region of NLR-C (Fig. 3b). Phylogenetic analysis of NLR-B and NLR-C proteins along with other NB-LRR proteins showed that NLR-B and NLR-C are structurally related to RPM1, which recognizes the AvrRpm1 type III effector avirulence protein from P. syringae in Arabidopsis 47 . They were also closely related to NB-LRRs in Oryza sativa, which leads to lesions on the leaf blade and broad-range resistance to the fungal pathogen Pyricularia oryzae (syn. Magnaporthe oryzae) and the bacterial pathogen Xanthomonas oryzae pv. oryzae, together with strong growth reduction (OsRLR1) 48 , and rice blast resistance (Pid3) 49  Segregation analysis. We next tested whether OSR in NOG confers susceptibility in a dominant fashion as reported previously. Followed by confirmation of the F 1 genotype by PCR, we investigated OPS in each F 1 plant (Fig. 4). The results showed that the response to fenitrothion at the seedling stage was somewhat less severe and variable, whereas lesions were clearly observed on the mature leaves in all F 1 plants. Although the precise reason for the unstable appearance of lesions at the seedling stage was unclear, it was apparent that all F 1 plants exhibited OPS; based on these observations, we concluded that OPS is semi-dominant.
To examine if the observed polymorphisms mentioned previously were indeed linked to the OPS phenotype, segregation analysis was performed. Our survey of RIL individuals showing intermediate OPS scorings in the F 8 population found that RIL25 showed segregation of the OSR locus. Therefore, F 9 progeny derived from an F 8 plant heterozygous for the NB-LRR cluster was subjected to further segregation analysis. A total of 86 plants of RIL25 from the F 9 generation were treated with fenitrothion at the heading stage in a greenhouse. Phenotyping of these plants indeed showed the segregation (Fig. 5a). Linkage analysis with genotyping data with NLR-A and NLR-C indicated that OSR segregated in a Mendelian fashion (Fig. 5b,c). Most importantly, co-segregation between OPS and NOG genotypes was found (Fig. 5d). These results strongly supported the notion that the OSR locus is semi-dominant and linked with NLR-B and NLR-C.

Transcript accumulation of NB-LRR genes indicates altered expression in NLR-C.
We performed semi-quantitative RT-PCR using gene-specific primers in the segregating RIL25 population along with the parental lines (Fig. 6a). RNA was extracted from mature leaves, either before or after fenitrothion treatment, and subjected to RT-PCR. For NLR-A, its transcript levels appeared to be extremely low and were not detectable in any samples irrespective of the presence (BTx623) or absence (NOG) of the gene. For NLR-B, we observed  www.nature.com/scientificreports/ weak signals in all samples, and the expression level did not respond to fenitrothion treatment. In contrast, NLR-C was shown to be expressed at a substantial level and to be detectable by our RT-PCR analyses (30 and 35 cycles). Intriguingly, NLR-C was constitutively expressed in NOG regardless of fenitrothion treatment, whereas no signals corresponding to NLR-C transcripts were detected in BTx623. Consistent with this observation, RIL25 F 9 segregants heterozygous for OSR and exhibiting OPS phenotype accumulated NLR-C mRNAs, whereas segregants homozygous for OSR and exhibiting resistance to fenitrothion was missing NLR-C transcripts (Fig. 6a).

Functionality of NLR-C in NOG was accounted for by the presence of the promoter.
To account for the differential expression of NLR-C, we reasoned that the 593-bp insertion present in NOG may positively affect transcription. This insertion is located 138 bp upstream of the ATG initiation codon of NLR-C extending towards the 5′ direction (Fig. 6b), suggesting that this insertion generates transcription initiation and/or part of 5′-untranslated region (UTR). Given that no transcript accumulated in BTx623, we postulated that the insertion contains a promoter activity and transcription initiates from this region. To examine this possibility, 5′-rapid amplification of cDNA ends (RACE) was performed with total RNA extracted from NOG and BTx623 with specific primers to detect the 5′ end of NLR-C transcripts ( Supplementary Fig. S5a). The result showed that, as expected, the amplified band corresponding to the 5′ end was detectable only in NOG ( Supplementary Fig. S5b).
Sequencing of the amplified fragment demonstrated that the 5′ end lies in A at 365-bp upstream of ATG initiation codon ( Supplementary Fig. S5c). Consequently NLR-C mRNA possesses a 365-bp 5′-UTR, within which no additional ORF was found. Furthermore, searches for core promoter elements in O. sativa in the database (http:// bioin forma tics. psb. ugent. be/ webto ols/ plant care/ html) revealed that a putative promoter sequence, TAT ATA ACA ATA TAT GTA CAAAA, exists 162-bp upstream of the start-site of NLR-C transcripts, which is also included in the 593-bp insertion (Fig. 6). Based on these results, we concluded that BTx623 has a null mutation in NLR-C due to the loss of a functional promoter, and that NLR-C is responsible for OPS in NOG.

Discussion
Sensitivity to organophosphate pesticides has been reported in sorghum by several research groups, as organophosphate insecticide reaction (opr) 42,43 and resistance to chemical burning (rcb) 45 . Although they mapped these loci using conventional DNA markers with different mapping populations, their location on chromosome 5 was suggested to represent the same locus 44 2020) investigated organophosphate-sensitive reaction (osr) using a sensitive inbred Nakei MS3B and raised the possibility that the same NB-LRR gene cluster is indeed responsible for OPS 46 . Based on the differential accumulation of transcripts in these three genes (termed A, B, and C in this study), NLR-C was supposed to be the major gene controlling OPS. The existence of amino-acid substitutions between Nakei MS3B and the resistant lines (BTx623 and Greenleaf) was also reported for NLR-C. Our current mapping of OPS observed in NOG was consistent with these previous reports. Detailed comparison of the nucleotide sequence along with transcript accumulation between BTx623 and NOG led us to conclude that NLR-C is the major gene. Although these reports identified NLR-C as a candidate gene, a discrepancy exists over the dominance of OPS. In most of studies, OPS was shown to be dominant. However, only Kawahigashi et al. (2020) reported OPS in Nakei MS3B as recessive 46 . As for the expression of NLR-C, they showed that resistant BTx623 had no detectable transcripts, whereas sensitive Nakei MS3B had substantial levels of NLR-C transcripts, strongly arguing against the dominant role of resistance in BTx623. In fact, we demonstrated here that BTx623 has a null mutation due to the loss of promoter activity, which cannot cause a dominant effect on resistance. One possibility to explain this inconsistency may be the unstable appearance of OPS in the heterozygotes. In our study, a careful inspection of F 1 plants showed that the lesion owing to OPS was weak and observed unstably at the seedling stage, whereas OPS became rather apparent in mature leaves (Fig. 4). These observations implied that the expression of OPS may be influenced developmentally, although the precise reason for this instability was unclear. We thus consider that re-evaluation is necessary to confirm whether OPS in Nakei MS3B is dominant.
Given the dominant effect of OPS in NOG, we further characterized the NB-LRR gene cluster. We excluded NLR-A because it is missing in NOG. Therefore, we considered that NLR-B and NLR-C are responsible for OPS. As for NLR-B, transcripts accumulated both in NOG and BTx623, whereas two amino acid substitutions in NOG (I289V and S650R) resides in the NB and LRR domains, respectively, and may account for the dominant effect of OPS. As for NLR-C, we have presented compelling evidence that the 593-bp deletion in BTx623 leads to the loss of promoter activity and a null mutation. Thus, a 593-bp insertion in the promoter region of NLR-C may be a determinant of the OPS phenotype in NOG. To test if NLR-C is involved and has dominant role in OPS, we performed heterologous complementation assays using agrobacterium infiltration in Nicotiana benthamiana ( Supplementary Fig. S6). Overexpression of NLR-C and concomitant treatment with fenitrothion, however, did not result in necrotic lesions, as opposed to the control, suggesting that OPS is not simply conferred by NB-LRR and may require species-specific signaling cascades to respond to organophosphate pesticides. An alternative possibility is the requirement of both NLR-B and NLR-C to drive OPS.
OPS has been extensively characterized in tomato 25,27,50 . The Fen gene, which corresponds to the organophosphate pesticide fenitrothion sensitivity phenotypes, encodes a serine-threonine protein kinase 28 . The Fen and Pto genes share 87% amino acid similarity. Fen kinase is needed for fenthion sensitivity. Reports have shown that Fen stimulates organophosphate pesticide fenthion-inducible signaling and interacts with the N-terminal domain of a NB-LRR protein Prf 29 . Prf is indispensable for fenthion sensitivity, but the role of Fen and its association with www.nature.com/scientificreports/ Prf 51 in fenthion sensitivity are poorly understood. Another example of an NB-LRR responding to chemicals is At5g46520 in Arabidopsis, which responds to accession-specific root growth arrest with DFPM 32,33 . DFPM was shown to be related to several abscisic acid (ABA) responses, including rapid disruption of ABA-induced stomatal closing and ABA activation of guard cell anion channels. Detailed analyses have uncovered that DFPM stimulates an ETI and results in rapid disruption of ABA signal transduction. A genetic variant of the NB-LRR gene in the Arabidopsis Columbia-0 accession is responsive specifically to DFPM by generating primary root meristem arrest. Although there has been extensive research on R genes that defense against microbial pathogens, there are few studies on the toxic reaction in plants from organophosphate chemicals. In sorghum, the resistance to a necrotrophic fungus Bipolaris sorghicola, controlled by ds1 encoding an NB-LRR has been reported 52 . Because OSR and ds1 are distantly mapped on chromosome 5, the OSR locus is unlikely to be involved in the resistance to B. sorghicola 53 . Future study is required to test if any of the NLR genes in the OSR locus is associated with pathogens, similarly to Pto/Fen in tomato. NLR-C shows similarity to the NB-LRR protein Arabidopsis RPM1 (Supplementary Fig. S4). Notably, a mutation in one of the rice RPM1 homologs OsRLR1 resulting from ethyl methane sulfonate treatment led to spontaneous hypersensitive response-like lesions with strong growth reduction, which was similar to the OPS phenotype 48 . Based on the results in Supplementary Fig. S5, it appears that NLR-C indirectly recognizes fenitrothion. RPM1 indirectly recognizes the Pseudomonas syringae effectors AvrB and AvrRpm1 through an Arabidopsis RIN4 protein 54 . AvrB and AvrRpm1 phosphorylate RIN4, and RPM1 monitors this phosphorylation and causes ETI. It is possible that NLR-C employs a host protein(s) to sense fenitrothion. Alternatively, evidence has been accumulating that sensor and helper NB-LRR pairs work together to perceive pathogen effectors and induce ETI 55 . Conceptually, it is possible that NLR-B and NLR-C act in a complex with the organophosphate molecules or act downstream as part of the OSR signaling pathway.
Our previous analysis of QTLs related to biomass in the same population did not find a QTL on chromosome 5. Therefore, its impact on biomass is unlikely. However, the OPS phenotype might decrease production in sorghum upon pesticide application, and our understanding of the OSR locus presented in this study is important in future sorghum breeding. In particular, unstable appearance of OPS along with its dominance over resistance should be specially taken into consideration because it potentially affects F 1 hybrids commonly used for commercial sorghum production. Our preliminary evaluation of OPS among the sorghum core collection used in our previous study indicated that most sorghum inbred lines are free from OPS. Nevertheless, two additional lines (IS11331 and Manfredi Cholila) were shown to have the same OSR haplotype as NOG and exhibit OPS at the seedling stage ( Supplementary Fig. S7). Our previous admixture analysis categorized the inbred lines into three groups, where NOG belonged to Group II that represented Asian accessions 39 . While both NOG and Manfredi Cholila were clustered in Group II, IS11331 belonged to Group I. Although further study is required, these results may implicate that the rare OPS originate from multiple genetic backgrounds.
In conclusion, the presented results along with previous reports strongly suggest that the OSR locus corresponds to the NB-LRR gene cluster, particularly to NLR-B and NLR-C. OSR is dominant with the unstable appearance of OPS in the F 1 generation, implicating particular caution in securing proper leaf longevity in breeding programs. Further investigations should allow us to understand the occurrence of OPS and its generic variation among sorghum cultivars, which may be related to diversification of the NB-LRR gene families.

Methods
Plant materials. Two parental lines, BTx623 and NOG, and the RIL population were established as described in our previous study 39 . All plant materials including each RIL generation were grown, harvested, and stored at the Institute of Plant Science and Resources (IPSR), according to the institutional guideline to handle plant materials. A genetic map was constructed using RAD-seq, and the genotyping data of 213 individuals were constructed in the F 6 population. In this study, the F 9 , F 10 , and F 11 populations were subjected to phenotyping of pesticide resistance, followed by QTL analysis. Phenotyping was carried out in multiple years with one replicate for each cultivation. Plants were germinated in small trays filled with vermiculite for approximately 10-14 days, subsequently transplanted to 30-cm diameter pots filled with soil from IPSR field. A single RIL line (RIL25) segregating for OPS was obtained by screening the F 8 population. Seeds from RIL25 were harvested from an F 8 individual that was heterozygous to OSR (confirmed by PCR), and the resulting F 9 segregants were subjected to segregation analysis. F 1 seeds were generated by crossing NOG with BTx623 with manual emasculation.  , Kurashiki city, Okayama, Japan. Seedlings at the fourth-leaf stage were sprayed uniformly with 500 × diluted fenitrothion, malathion or acephate (Sumitomo Chemical Garden Products INC, Japan), and the phenotype was scored visually (from 1 to 5) at 7 days after the pesticide treatment. A score of 1 indicated essentially no leaf death, a score of 3 indicated approximately 50% of the leaf area was dead, and a score of 5 indicated 100% seedling (leaves and stem) death. The sensitive lines showed red-brown spots and necrotic lesions throughout their growth, and the resistant lines maintained greener leaf area. Flag leaves were treated with fenitrothion from F 1 plants and the RIL25-F 9 segregating population at the heading stage, and the greenness of plants was estimated visually 7 days after fenitrothion application. Moreover, evaluation of the greenness trait with fenitrothion treatment was performed for each RIL.
QTL analysis. Genotype data obtained from RAD-seq in the F 6 population was used to perform QTL analysis in this study. A total of 3710 SNPs distributed across the genome were obtained. A dense linkage map with 3710 markers spanning a distance of 644.8 cM on all chromosomes, with an average and maximum spacing between markers of 0.2 cM and 7.1 cM, respectively, was developed previously. Genotype probabilities were calculated using the calc.genoprobability function with a step size of 1 cM and an assumed genotyping error probability of 0.05 using the Kosambi map function as implemented in the R/qtl package 56,57 . QTL analysis was performed using the CIM function of the R/qtl package with the Haley-Knott regression method 58 . Linkage analysis was performed using the R/qtl package in R version 3.4.2. The LOD significance threshold for detecting QTLs was calculated by performing 1000 iterations using the R/qtl permutation test.  Supplementary Information 2. (b) F 1 plants (#1-#8) were grown from seedling to mature stages along with NOG and BTx623, and fenitrothion was spotted onto the fourth or fifth leaves at the seedling stage as illustrated (lower panels. Scale bars; 1 cm) or sprayed onto mature leaves (upper panels, Scale bars; 0.6 cm). www.nature.com/scientificreports/  5′-RACE analysis of candidate gene. The 5′ RACE experiment was performed using a 5-Full RACE Core Set Kit (TAKARA, Japan) in accordance with the manufacturer's instructions. Fifteen microliters of ligated cDNA was then used as a template for nested PCR using KOD FX Neo polymerase (TOYOBO, Japan) with two sets of primers, the outside set was sense primer_1 and antisense primer_1, and the inside set is sense primer_2 and antisense primer_2. The PCR reaction mixture was incubated for 2 min at 94 °C followed by 30 amplification cycles, comprising denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s and extension at 68 °C for 30 s. The reaction was extended for another 10 min at 68 °C to ensure full extension. Primers used in this study are listed in Supplementary Table S2.

Transient expression of NLR-C in Nicotiana benthamiana.
Agroinfiltration of N. benthamiana was performed as described previously 62 . A. tumefaciens strain GV3101, carrying NLR-C, Pit D485V, or GFP was used to infiltrate leaves of 5-week-old N. benthamiana plants. p19 silencing suppressor was used to enhance gene expression. Each agrobacterium culture was resuspended in buffer containing 10 mM MgCl 2 , 10 mM MES, pH 5.6, and 150 µM acetosyringone, and incubated at room temperature for 2-3 h before infiltration. After incubation for 24 h, fenitrothion (1:7500 dilution) or distilled water were infiltrated into the same spots as used for agroinfiltration. The photosynthetic capacity was measured at 3 days post-inoculation (dpi) using an imaging fluorimeter (FluorCam 800MF, photon systems instruments). Infiltrated plants were kept at 22 °C. Photographs were taken at 17 days post-inoculation (dpi) for the assessment of cell death. Figure 6. Expression of NLR genes in OSP and null mutation in NLR-C. (a) Transcript accumulation of the three NB-LRR genes (NLR-A, NLR-B, and NLR-C) estimated using semi-quantitative RT-PCR. Expression of NLR-A, NLR-B, and NLR-C in leaves. Accumulation of NLR-C transcripts coincides with the presence of the fenitrothion-sensitive (S) NOG genotype (NOG, RIL25-78, − 80, and − 81), whereas no transcript was detected in BTx623 and the resistant (R) line (RIL25-79). '+' and '−' indicate gene expression analysis in the presence or absence of organophosphate pesticide (OP), respectively. The PP2A gene was used as an internal control. The original gel images are provided in Supplementary Information 2. (b) Schematic diagram of the NLR-C coding region and its 5' upstream region. NLR-C consists of a single exon (top), and the closeup view of the 593-bp insertion specific to NOG is shown below. Putative transcription start site (TSS, indicated by an arrow) is located 227 bp upstream from the 3′ end of the insertion. The nucleotide sequence of the insertion is shown below the diagram, where the putative TSS determined by 5′ RACE and the TATA-box sequence are indicated. A schematic diagram of the insertion region is shown, which contains the TSS and putative TATA-box, upstream NLR-C gene in NOG genetic background.