Novel insecticidal proteins from ferns resemble insecticidal proteins from Bacillus thuringiensis.

Lepidopterans affect crop production worldwide. The use of transgenes encoding insecticidal proteins from Bacillus thuringiensis (Bt) in crop plants is a well-established technology that enhances protection against lepidopteran larvae. Concern about widespread field-evolved resistance to Bt proteins has highlighted an urgent need for new insecticidal proteins with different modes or sites of action. We discovered a new family of insecticidal proteins from ferns. The prototype protein from Pteris species (Order Polypodiales) and variants from two other orders of ferns, Schizaeales and Ophioglossales, were effective against important lepidopteran pests of maize and soybean in diet-based assays. Transgenic maize and soybean plants producing these proteins were more resistant to insect damage than controls. We report here the crystal structure of a variant of the prototype protein to 1.98 Å resolution. Remarkably, despite being derived from plants, the structure resembles the 3-domain Cry proteins from Bt but has only two out of three of their characteristic domains, lacking the C-terminal domain which is typically required for their activities. Two of the fern proteins were effective against strains of fall armyworm that were resistant to Bt 3-domain Cry proteins Cry1Fa or Cry2A.127. This therefore represents a novel family of insecticidal proteins that have the potential to provide future tools for pest control.

A pigeonpea gene confers resistance to Asian soybean rust in soybean.

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most economically important crop diseases, but is only treatable with fungicides, which are becoming less effective owing to the emergence of fungicide resistance. There are no commercial soybean cultivars with durable resistance to P. pachyrhizi, and although soybean resistance loci have been mapped, no resistance genes have been cloned. We report the cloning of a P. pachyrhizi resistance gene CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) from pigeonpea (Cajanus cajan) and show that CcRpp1 confers full resistance to P. pachyrhizi in soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement.

Identification of Phakopsora pachyrhizi Candidate Effectors with Virulence Activity in a Distantly Related Pathosystem.

Phakopsora pachyrhizi is the causal agent of Asian Soybean Rust, a disease that causes enormous economic losses, most markedly in South America. P. pachyrhizi is a biotrophic pathogen that utilizes specialized feeding structures called haustoria to colonize its hosts. In rusts and other filamentous plant pathogens, haustoria have been shown to secrete effector proteins into their hosts to permit successful completion of their life cycle. We have constructed a cDNA library from P. pachyrhizi haustoria using paramagnetic bead-based methodology and have identified 35 P. pachyrhizi candidate effector (CE) genes from this library which are described here. In addition, we quantified the transcript expression pattern of six of these genes and show that two of these CEs are able to greatly increase the susceptibility of Nicotiana benthamiana to Phytophthora infestans. This strongly suggests that these genes play an important role in P. pachyrhizi virulence on its hosts.

Development of genomic SSR markers for fingerprinting lettuce (Lactuca sativa L.) cultivars and mapping genes.

BACKGROUND: Lettuce (Lactuca sativa L.) is the major crop from the group of leafy vegetables. Several types of molecular markers were developed that are effectively used in lettuce breeding and genetic studies. However only a very limited number of microsattelite-based markers are publicly available. We have employed the method of enriched microsatellite libraries to develop 97 genomic SSR markers. RESULTS: Testing of newly developed markers on a set of 36 Lactuca accession (33 L. sativa, and one of each L. serriola L., L. saligna L., and L. virosa L.) revealed that both the genetic heterozygosity (UHe = 0.56) and the number of loci per SSR (Na = 5.50) are significantly higher for genomic SSR markers than for previously developed EST-based SSR markers (UHe = 0.32, Na = 3.56). Fifty-four genomic SSR markers were placed on the molecular linkage map of lettuce. Distribution of markers in the genome appeared to be random, with the exception of possible cluster on linkage group 6. Any combination of 32 genomic SSRs was able to distinguish genotypes of all 36 accessions. Fourteen of newly developed SSR markers originate from fragments with high sequence similarity to resistance gene candidates (RGCs) and RGC pseudogenes. Analysis of molecular variance (AMOVA) of L. sativa accessions showed that approximately 3% of genetic diversity was within accessions, 79% among accessions, and 18% among horticultural types. CONCLUSIONS: The newly developed genomic SSR markers were added to the pool of previously developed EST-SSRs markers. These two types of SSR-based markers provide useful tools for lettuce cultivar fingerprinting, development of integrated molecular linkage maps, and mapping of genes.

A real-time PCR assay for detection and quantification of Verticillium dahliae in spinach seed.

Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.

Quantitative resistance to late blight from Solanum berthaultii cosegregates with R(Pi-ber): insights in stability through isolates and environment.

Genetic resistance is a valuable tool in the fight against late blight of potatoes but little is known about the stability and specificity of quantitative resistance including the effect of defeated major resistance genes. In the present study we investigated the effect of different isolates of Phytophthora infestans on the mode of action of R(Pi-ber), an R-gene originating from Solanum berthaultii. The experiments were conducted on progenies derived from two reciprocal inter-specific backcrosses of Solanum tuberosum and S. berthaultii. The plant-pathogen interaction was tested in diverse environments including field, greenhouse and growth chamber conditions. The R(Pi-ber) gene provided complete resistance against a US8 isolate of P. infestans in all trials. When isolates compatible with R(Pi-ber) were used for inoculation, a smaller, but significant resistance effect was consistently detected in the same map position as the R-gene. This indicates that this R-gene provides a residual resistance effect, and/or that additional resistance loci are located in this genomic region of chromosome X. Additional quantitative resistance loci (QRL) were identified in the analyzed progenies. While some of the QRL (such as those near TG130 on chromosome III) were effective against several isolates of the pathogen, others were isolate specific. With a single exception, the S. berthaultii alleles were associated with a decrease in disease severity. Resistance loci reported in the present study co-locate with previously reported R-genes and QRL to P. infestans and other pathogens.