Targeting the Highly Conserved 3' Untranslated Region of Iris Severe Mosaic Virus for Sensitive Monitoring of the Disease Prevalence in Iris Production.

Iris severe mosaic virus (ISMV, Potyviridae) can threaten the sustainability of iris production and the marketability of the plants. Effective intervention and control strategies require rapid and early detection of viral infections. The wide range of viral symptoms, from asymptomatic to severe chlorosis of the leaves, renders diagnosis solely based on visual indicators ineffective. A nested PCR-based diagnostic assay was developed for the reliable detection of ISMV in iris leaves and in rhizomes. Considering the genetic variability of ISMV, two primer pairs were designed to detect the highly conserved 3' untranslated region (UTR) of the viral genomic RNA. The specificity of the primer pairs was confirmed against four other potyviruses. The sensitivity of detection was enhanced by one order of magnitude using diluted cDNA and a nested approach. Nested PCR facilitated detecting ISMV on field-grown samples beyond the capabilities of a currently available immunological test and in iris rhizome, which would facilitate ensuring clean stock is planted. This approach dramatically improves the detection threshold of ISMV on potentially low virus titer samples. The study provides a practical, accurate, and sensitive tool for the early detection of a deleterious virus that infects a popular ornamental and landscape plant.

An atypical N-ethylmaleimide sensitive factor enables the viability of nematode-resistant Rhg1 soybeans.

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 Heterodera glycines 1" (Rhg1) locus of soybean (Glycine max) confers resistance to soybean cyst nematode, a highly damaging soybean pest. Rhg1 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 NSF allele (Rhg1-associated NSF on chromosome 07; NSFRAN07 ) in Rhg1+ germplasm. NSFRAN07 protein modeling to mammalian NSF/α-SNAP complex structures indicated that at least three of the five NSFRAN07 polymorphisms reside adjacent to the α-SNAP binding interface. NSFRAN07 exhibited stronger in vitro binding with Rhg1 resistance-type α-SNAPs. NSFRAN07 coexpression in planta was more protective against Rhg1 α-SNAP cytotoxicity, relative to WT NSFCh07 Investigation of a previously reported segregation distortion between chromosome 18 Rhg1 and a chromosome 07 interval now known to contain the Glyma.07G195900 NSF gene revealed 100% coinheritance of the NSFRAN07 allele with disease resistance Rhg1 alleles, across 855 soybean accessions and in all examined Rhg1+ progeny from biparental crosses. Additionally, we show that some Rhg1-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 Rhg1-related resistance in plants will require a compatible NSF partner for the resistance-conferring α-SNAP.

Structurally distinct Arabidopsis thaliana NLR immune receptors recognize tandem WY domains of an oomycete effector.

Nucleotide-binding leucine-rich repeat (NB-LRR, or NLR) receptors mediate pathogen recognition. The Arabidopsis thaliana NLR RPP1 recognizes the tandem WY-domain effector ATR1 from the oomycete Hyaloperonospora arabidopsidis through direct association with C-terminal LRRs. We isolated and characterized homologous NLR genes RPP1-EstA and RPP1-ZdrA from two Arabidopsis ecotypes, Estland (Est-1) and Zdarec (Zdr-1), responsible for recognizing a novel spectrum of ATR1 alleles. RPP1-EstA and -ZdrA encode nearly identical NLRs that are phylogenetically distinct from known immunity-activating RPP1 homologs and possess greatly expanded LRR domains. Site-directed mutagenesis and truncation analysis of ATR1 suggests that these homologs recognize a novel surface of the 2(nd) WY domain of ATR1, partially specified by a C-terminal region of the LRR domain. Synteny comparison with RPP1 loci involved in hybrid incompatibility suggests that these functions evolved independently. Closely related RPP1 homologs have diversified their recognition spectra through LRR expansion and sequence variation, allowing them to detect multiple surfaces of the same pathogen effector. Engineering NLR receptor specificity may require a similar combination of repeat expansion and tailored amino acid variation.

Detection of rare nematode resistance Rhg1 haplotypes in Glycine soja and a novel Rhg1 α-SNAP.

This study pursued the hypothesis that wild plant germplasm accessions carrying alleles of interest can be identified using available single nucleotide polymorphism (SNP) genotypes for particular alleles of other (unlinked) genes that contribute to the trait of interest. The soybean cyst nematode (SCN, Heterodera glycines [HG]) resistance locus Rhg1 is widely used in farmed soybean [Glycine max (L.) Merr.]. The two known resistance-conferring haplotypes, rhg1-a and rhg1-b, typically contain three or seven to 10 tandemly duplicated Rhg1 segments, respectively. Each Rhg1 repeat carries four genes, including Glyma.18G022500, which encodes unusual isoforms of the vesicle-trafficking chaperone α-SNAP. Using SoySNP50K data for NSFRAN07 allele presence, we discovered a new Rhg1 haplotype, rhg1-ds, in six accessions of wild soybean, Glycine soja Siebold & Zucc. (0.5% of the ∼1,100 G. soja accessions in the USDA collection). The α-SNAP encoded by rhg1-ds is unique at an important site of amino acid variation and shares with the rhg1-a and rhg1-b α-SNAP proteins the traits of cytotoxicity and altered N-ethylmaleimide sensitive factor (NSF) protein interaction. Copy number assays indicate three repeats of rhg1-ds. G. soja PI 507613 and PI 507623 exhibit resistance to HG type 2.5.7 SCN populations, in part because of contributions from other loci. In a segregating F2 population, rhg1-b and rhg1-ds made statistically indistinguishable contributions to resistance to a partially virulent HG type 2.5.7 SCN population. Hence, the unusual multigene copy number variation Rhg1 haplotype was present but rare in ancestral G. soja and was present in accessions that offer multiple traits for SCN resistance breeding. The accessions were initially identified for study based on an unlinked SNP.

The rhg1-a (Rhg1 low-copy) nematode resistance source harbors a copia-family retrotransposon within the Rhg1-encoded α-SNAP gene.

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 rhg1-a 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 Rhg1WT (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-SNAPRhg1LC, 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 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.