N-ethylmaleimide sensitive factor (NSF) and α-soluble NSF attachment protein (α-SNAP) are essential eukaryotic housekeeping proteins that cooperatively function to sustain vesicular trafficking. The "resistance to 1" () locus of soybean () confers resistance to soybean cyst nematode, a highly damaging soybean pest. loci encode repeat copies of atypical α-SNAP proteins that are defective in promoting NSF function and are cytotoxic in certain contexts. Here, we discovered an unusual allele (-associated NSF on chromosome 07; ) in germplasm. NSF protein modeling to mammalian NSF/α-SNAP complex structures indicated that at least three of the five NSF polymorphisms reside adjacent to the α-SNAP binding interface. NSF exhibited stronger in vitro binding with resistance-type α-SNAPs. NSF coexpression was more protective against α-SNAP cytotoxicity, relative to WT NSF Investigation of a previously reported segregation distortion between chromosome 18 and a chromosome 07 interval now known to contain the NSF gene revealed 100% coinheritance of the allele with disease resistance alleles, across 855 soybean accessions and in all examined progeny from biparental crosses. Additionally, we show that some-mediated resistance is associated with depletion of WT α-SNAP abundance via selective loss of WT α-SNAP loci. Hence atypical coevolution of the soybean SNARE-recycling machinery has balanced the acquisition of an otherwise disruptive housekeeping protein, enabling a valuable disease resistance trait. Our findings further indicate that successful engineering of -related resistance in plants will require a compatible NSF partner for the resistance-conferring α-SNAP.

Toxigenic Clostridium sordellii causes uncommon but highly lethal infections in humans and animals. Recently, an increased incidence of C. sordellii infections has been reported in women undergoing obstetric interventions. Pathogenic strains of C. sordellii produce numerous virulence factors, including sordellilysin, phospholipase, neuraminidase, and two large clostridial glucosylating toxins, TcsL and TcsH. Recent studies have demonstrated that TcsL toxin is an essential virulence factor for the pathogenicity of C. sordellii. In this study, we identified and characterized TcsR as the toxin gene (tcsL) regulator in C. sordellii. Highthroughput sequencing of two C. sordellii strains revealed that tcsR lies within a genomic region that encodes TcsL, TcsH, and TcsE, a putative holin. By using ClosTron technology, we inactivated the tcsR gene in strain ATCC 9714. Toxin production and tcsL transcription were decreased in the tcsR mutant strain. However, the complemented tcsR mutant produced large amounts of toxins, similar to the parental strain. Expression of the Clostridium difficile toxin gene regulator tcdR also restored toxin production to the C. sordellii tcsR mutant, showing that these sigma factors are functionally interchangeable. Clostridium sordellii, an anaerobic, Gram-positive, spore-forming bacterium, is a common inhabitant of soil and the animal gastrointestinal tract. Virulent strains of C. sordellii are recognized as the causative agents of a broad spectrum of human diseases, including myonecrosis, uterine infections, and sepsis. C. sordellii is also known to cause lethal infections in several animal species, including sheep, foals, and lambs (1-4). Recently, fatal cases of C. sordellii endometritis following medical abortions caused by mifepristone-misoprostol combinations have been reported (5). It has been suggested that mifepristone-misoprostol may facilitate colonization of C. sordellii in uterine tissue, trigger toxin expression, and induce hypotension and systemic shock by deregulating the host's immune response (6).Pathogenic C. sordellii strains produce up to seven identified exotoxins (7). Of these, the two major toxins, lethal toxin (TcsL) and the hemorrhagic toxin (TcsH), are regarded as major virulence factors (8, 9). The lethal toxin produced by C. sordellii was shown to evoke enteritis in animals and proved essential for the virulence of C. sordellii (9, 10). TcsH and TcsL are members of the large clostridial cytotoxin (LCC) family, with predicted molecular masses of 300 kDa and 250 kDa, respectively (8, 9). The C. sordellii toxins were reported to be similar to Clostridium diffcile toxins A and B, both in terms of biological activity and antigenicity (11). To date, only the TcsL-encoding gene has been sequenced; it was found to be 76% identical to the C. difficile toxin B gene (tcdB).In this study, we sequenced two C. sordellii strains, ATCC 9714 and VPI 9048, by high-throughput techniques, and we identified many open reading frames (ORFs) surrounding the tcsL gene. Consistent with pr...

Soybean growers widely use the R esistance to H eterodera g lycines 1 ( Rhg1 ) locus to reduce yield losses caused by soybean cyst nematode (SCN). Rhg1 is a tandemly repeated four gene block. Two classes of SCN resistance‐conferring Rhg1 haplotypes are recognized: rhg1‐a (“Peking‐type,” low‐copy number, three or fewer Rhg1 repeats) and rhg1‐b (“PI 88788‐type,” high‐copy number, four or more Rhg1 repeats). The rhg1‐a and rhg1‐b haplotypes encode α‐SNAP (alpha‐ S oluble N SF A ttachment P rotein) variants α‐SNAP Rhg1 LC and α‐SNAP Rhg1 HC, respectively, with differing atypical C‐terminal domains, that contribute to SCN resistance. Here we report that rhg1‐a soybean accessions harbor a copia retrotransposon within their Rhg1 Glyma.18G022500 (α‐SNAP‐encoding) gene. We termed this retrotransposon “ RAC, ” for R hg1 a lpha‐SNAP c opia. Soybean carries multiple RAC ‐like retrotransposon sequences. The Rhg1 RAC insertion is in the Glyma.18G022500 genes of all true rhg1‐a haplotypes we tested and was not detected in any examined rhg1‐b or Rhg1 WT (single‐copy) soybeans. RAC is an intact element residing within intron 1, anti‐sense to the rhg1‐a α‐SNAP open reading frame. RAC has intrinsic promoter activities, but overt impacts of RAC on transgenic α‐SNAP Rhg1 LC mRNA and protein abundance were not detected. From the native rhg1‐a RAC + genomic context, elevated α‐SNAP Rhg1 LC protein abundance was observed in syncytium cells, as was previously observed for α‐SNAP Rhg1 HC (whose rhg1‐b does not carry RAC ). Using a SoySNP50K SNP corresponding with RAC presence, just ~42% of USDA accessions bearing previously identified rhg1‐a SoySNP50K SNP signatures harbor the RAC insertion. Subsequent analysis of several of these putative rhg1‐a accessions lacking RAC revealed that none encoded α‐SNAP Rhg1 LC , and thus, they are not rhg1‐a . ...

Soybean growers widely use the Resistance to Heterodera glycines 1 (Rhg1) locus to reduce yield losses caused by soybean cyst nematode (SCN). Rhg1 is a tandemly repeated four gene block. Two classes of SCN resistance-conferring Rhg1 haplotypes are recognized: rhg1-a ("Peking-type," low-copy number, three or fewer Rhg1 repeats) and rhg1-b ("PI 88788-type," high-copy number, four or more Rhg1 repeats). The rhg1a and rhg1-b haplotypes encode α-SNAP (alpha-Soluble NSF Attachment Protein) variants α-SNAP Rhg1 LC and α-SNAP Rhg1 HC, respectively, with differing atypical C-terminal domains, that contribute to SCN resistance. Here we report that rhg1-a soybean accessions harbor a copia retrotransposon within their Rhg1 Glyma.18G022500 (α-SNAP-encoding) gene. We termed this retrotransposon "RAC," for Rhg1 alpha-SNAP copia. Soybean carries multiple RAC-like retrotransposon sequences. The Rhg1 RAC insertion is in the Glyma.18G022500 genes of all true rhg1-a haplotypes we tested and was not detected in any examined rhg1-b or Rhg1 WT (single-copy) soybeans. RAC is an intact element residing within intron 1, anti-sense to the rhg1-a α-SNAP open reading frame. RAC has intrinsic promoter activities, but overt impacts of RAC on transgenic α-SNAP Rhg1 LC mRNA and protein abundance were not detected. From the native rhg1-a RAC + genomic context, elevated α-SNAP Rhg1 LC protein abundance was observed in syncytium cells, as was previously observed for α-SNAP Rhg1 HC (whose rhg1-b does not carry RAC). Using a SoySNP50K SNP corresponding with RAC presence, just ~42% of USDA accessions bearing previously identified rhg1-a SoySNP50K SNP signatures harbor the RAC insertion. Subsequent analysis of several of these putative rhg1-a accessions lacking RAC revealed that none encoded α-SNAP Rhg1 LC, and thus, they are not rhg1-a. rhg1-a haplotypes are of rising interest, with Rhg4, for combating SCN populations that exhibit increased virulence against the widely used 1997; Hussey, Boerma, Raymer, & Luzzi, 1991;Klepadlo et al., 2018;Vuong et al., 2015;Young, 1995)). However, the influence of all Rhg1 haplotype and/or allelic variation factors on SCN-resistance expression or plant yield is not yet fully understood.rhg1-b resistance. The present study reveals another unexpected structural feature of many Rhg1 loci, and a selectable feature that is predictive of rhg1-a haplotypes. K E Y W O R D Splant disease resistance, retrotransposon, Rhg1, soybean cyst nematode

Soybean cyst nematode is the most economically damaging pathogen of soybean and host resistance is a core management strategy. The SCN resistance QTL cqSCN-006, introgressed from the wild relative Glycine soja, provides intermediate resistance against nematode populations including those with increased virulence on the heavily used rhg1-b resistance locus. cqSCN-006 was previously fine-mapped to a genome interval on chromosome 15. The present study determined that Glyma.15G191200 at cqSCN-006, encoding a ɣ-SNAP (gamma-SNAP), contributes to SCN resistance. CRISPR/Cas9-mediated disruption of the cqSCN-006 allele reduced SCN resistance in transgenic roots. There are no encoded amino acid polymorphisms between resistant and susceptible alleles. However, other cqSCN-006-specific DNA polymorphisms in the Glyma.15G191200 promoter and gene body were identified, and we observed differing induction of ɣ-SNAP protein abundance at SCN infection sites between resistant and susceptible roots. We identified alternative RNA splice forms transcribed from the Glyma.15G191200 ɣ-SNAP gene and observed differential expression of the splice forms two days after SCN infection. Heterologous overexpression of ɣ-SNAPs in plant leaves caused moderate necrosis, suggesting that careful regulation of this protein is required for cellular homeostasis. Apparently, certain G. soja evolved quantitative SCN resistance through altered regulation of ɣ-SNAP. Previous work has demonstrated SCN resistance impacts of the soybean α-SNAP proteins encoded by Glyma.18G022500 (Rhg1) and Glyma.11G234500. The present study shows that a different type of SNAP protein can also impact SCN resistance. Little is known about ɣ-SNAPs in any system, but the present work suggests a role for ɣ-SNAPs during susceptible responses to cyst nematodes.