Overexpression of a Broccoli Defensin Gene BoDFN Enhances Downy Mildew Resistance

Plant defensins are small, basic cysteine-rich peptides that play important roles in disease resistance. A gene, designated BoDFN, was isolated from Brassica oleracea var. italica. Gene sequence has been submitted to NCBI with an accession no. of HQ436486. Complete coding sequence of BoDFN is 243 bp in length encoding 80 amino acids. Sequence comparison results showed that BoDFN shared high homology with those of crucifer plants and there were only few DNA base differences. RT-PCR results indicated an increase of gene expression in Hyaloperonospora parasitica infected leaves and revealed a significant increase at 24 and 36 h of treatment. A recombinant plasmid, named pBI121-BoDFN, was constructed and introduced into Agrobacterium tumefacien LBA4404. PCR screening for 65 regenerated plantlets, 17 positive plantlets were obtained, PCR screening results revealed that 17 out of 65 regenerated plantlets were positive. Disease resistant identification results indicated that all positive plants showed an increase in resistance to H. parasitica.

Cloning and Expression Analysis of Downy Mildew Resistance-Related cDNA Sequences in Melon

Melon downy mildew caused by Pseudoperonospora cubensis leads to significant losses in melon yields worldwide. Reverse-transcription Polymerase Chain Reaction （RT-PCR） was performed using cDNAs as templates from melon-Huangdanzi induced with fungus Pseudoperonospora cubensis, and degenerate primers designed based on the conserved amino acid sequences of known plant disease-resistance genes. A polymorphic cDNA fragment which we named rap-19 was cloned and sequenced. The Open Reading Frame （ORF） of this product comprised of 510 base pairs which encodes DNA or RNA-binding protein with 170 amino acids. The putative amino acid sequence of mp-19 appeared highly homologous with those of NBS-type resistant-genes isolated from other plants. Southern blot indicated that the melon genome contained more than 3 copies of rap-19. The obvious expression differences detected by semi-quantitative RT- PCR could be observed between resistant-line Huangdanzi and susceptible-line Jiashi after Pseudoperonospora cubensis infection, which implied that mp-19 gene may be related to the resistance of downy mildew in melon.

Breeding melon(Cucumis melo)with resistance to powdery mildew and downy mildew

Melon(Cucumis melo L.)production is often restricted by a plethora of pests and diseases,including powdery mildew and downy mildew caused respectively by the fungal species Podosphaera xanthii/Golovinomyces orontii and oomycete species Pseudoperonospora cubensis.Many efforts have been directed on identification of resistant sources by screening(wild)melon germplasm.In the current review,we summarized such efforts from various publications of the last 50 plus years.Resistance to powdery mildew has been identified in 239 melon accessions and downy mildew resistance in 452 accessions of both C.melo and the wild relative species C.figarei.Among the resistance sources,C.melo var.cantalupensis accessions PMR 45,PMR 5,PMR 6,and WMR 29 as well as C.melo var.momordica accessions PI 124111,PI 124112,and PI 414723 have been considered as the most valuable germplasm because multiple resistance genes have been identified from these accessions and are widely used in melon resistance breeding.Further genetic mapping in a number of resistant sources has enabled identification of 25 dominant genes,two recessive genes and seven QTLs conferring powdery mildew resistance,as well as eight dominant genes and 11 QTLs for downy mildew resistances.Based on the reported sequences of associated markers,we anchored physically(many of)these genes and QTLs to chromosomes of the melon cv.DHL92 genome.In addition to presenting a comprehensive overview on powdery mildew and downy mildew resistance in(wild)melon germplasm,we suggest strategies aiming at breeding melon with durable and broad-spectrum resistance to pathogens and pests.

Comparative proteomic profiles of resistant/susceptible cucumber leaves in response to downy mildew infection

Downy mildew is a serious disease in cucumber production worldwide,which is caused by Pseudoperonospora cubensis(Berk.&Curt.)Rostov.Underlying the mechanism of cucumber response to downy mildew infection is important for breeding improvement and production;however,the research remains largely elusive.A comparative proteomic approach was used to reveal the differential accumulation of the proteomes from leaves of cucumber(susceptible line and resistant line)that were inoculated with P.cubensis or not by two-dimensional electrophoresis and MALDI-TOF/TOF MS.A total of 76 protein spots were successfully identified with significant changes in abundance(>2-fold,P<0.05)in downy mildew infected or not leaves for the susceptible line and resistant line.By the functional annotation,these proteins were classified into 8 groups including photosynthesis(29%),energy and metabolism(29%),cell rescue and defense(17%),and protein biosynthesis,folding and degradation(13%),unclassified(7%),nucleotide metabolism(3%),signal transduction(1%)and cellular process(1%).Among the 17 differentially expressed proteins between the resistant and susceptible cucumber line,most of the protein spots were concentrated in cell rescue and defense(4)and energy and metabolism(4).Moreover,a schematic diagram containing majority of the metabolic pathways of cucumber leaves in response to downy mildew was proposed.This network revealed the positive effect of several functional components in cucumber seedlings’resistant to downy mildew such as accumulation of energy supply and resistance-related proteins,hastened protein metabolism and photorespiratory,inhibited photosynthesis,and triggered photosystem repair and programmed cell death.Taken together,these results have advanced a further understanding of the key metabolic pathways of cucumber resistance to downy mildew and pathogen control in the proteomic level.

Identification and Molecular Mapping of the Rs Dm R Locus Conferring Resistance to Downy Mildew at Seedling Stage in Radish(Raphanus sativus L.)

Downy mildew （DM）, caused by the fungus Peronospora parasitica, is a destructive disease of radish （Raphanus sativus L.） worldwide. Host resistance has been considered as an attractive and environmentally friendly approach to control the disease. However, the genetic mechanisms of resistance in radish to the pathogen remain unknown. To determine the inheritance of resistance to DM, F1, F2 and BC1F1 populations derived from reciprocal crosses between a resistant line NAU-dhp08 and a susceptible line NAU-qtbjq-06 were evaluated for their responses to DM at seedling stage. All F1 hybrid plants showed high resistance to DM and maternal effect was not detected. The segregation for resistant to susceptible individuals statistically iftted a 3：1 ratio in two F2 populations （F2（SR） and F2（RS））, and 1：1 ratio in two BC1F1 populations, indicating that resistance to DM at seedling stage in radish was controlled by a single dominant locus designated as RsDmR. A total of 1 972 primer pairs （1 036 SRAP, 628 RAPD, 126 RGA, 110 EST-SSR and 72 ISSR） were screened, and 36 were polymorphic between the resistant and susceptible bulks, and consequently used for genotyping individuals in the F2 population. Three markers （Em9/ga24370, NAUISSR826700 and Me7/em10400） linked to the RsDmR locus within a 10.0 cM distance were identiifed using bulked segregant analysis （BSA）. The SRAP marker Em9/ga24370 was the most tightly linked one with a distance of 2.3 cM to RsDmR. These markers tightly linked to the RsDmR locus would facilitate marker-assisted selection and resistance gene pyramiding in radish breeding programs.

Identification and Characterization of Simple Sequence Repeats (SSR) Markers Associated with Downy Mildew Resistance Locus “<i>RPV1</i>” in Grapes

The cultivation of grapes is severely impacted by the emergence of downy mildew (DM) disease which negatively affects quality and yield possibly resulting in heavy losses. Due to certain shortcomings in the usage of fungicides and the development of new cultivars by plant breeding, marker assisted selection (MAS) will be an efficient alternative method to introduce desired genes into the cultivated varieties in a short time period. The Simple sequence repeats (SSR) markers seem to be the most popular genetic marker of choice for MAS. In the present study, we identified 14 new SSR markers in RPV1 locus that are associated with downy mildew resistance in grapes. The characterization of the identified markers was carried out on the basis of various parameters such as types of repeat motifs, number of repeats, different classes and structure of microsatellites. Additionally, SSR genotyping in 56 different grape accessions was done to determine the susceptibility or resistance of these accessions to DM.

Preliminary Studies on Induced Resistance of Cucumber to Downy Mildew

1 mL of sporangial suspensions (5 x 10 5spporangia per milliliter) of Pseudoperonospora cubensis was droplet-inoculated on the surface of the second leaf of the plant grown in greenhouse ( inducing inoculatoin), then the lower surfaces of the third, the forth and the fifth leaves were uniformly sprayed with inoculum of the same fungi (5 x 10 4sporangia per milliliter, about 5 mL per plant) every 3 days interval (challenge inoculation). Plants were moistened at 18- 22 C for 18 h, then kept at room temperature (24 - 28 C) and supplemented with cool-white fluorescent lights. All three challenge leaves were collected after 7 days of challenge to measure the amount of sporulation and area of necrosis. Plants prior inoculated with P. cubensis were protected 38% (based on the area of necrosis) against disaesc caused by subsequent foliar challenge with the pathogen. Protective action was about 12% after 3 days, and maintained this level until 9 days, suddenly reached 34% after 12 days, and came to a maximum after 15 days, then dropped down slowly.