LecRK-1.9 Enhances Resistance of Solanaceous Plants Against Phytophthora Infestans
The potato cultivars that are susceptible to Phytophthora infestans were the end products of the potato plants bred for resistance and monitored for the gene called race-specific late blight resistance or R. The R gene belongs to the gene class called nucleotide binding site-leucine rich repeat (NB-LRR). This gene was observed to originate from Mexican wild potato plants like Solanum stoloniferum and S.demissum. But, prolonged resistance was not achieved as P.infestans could adapt rapidly to R genes. Recently, late blight disease was managed mostly by the fungicides which are known to show adverse effects on the environment and are expensive. Creating new resistant potato cultivars has become a major challenge in the recent potato breeding programs.
The proteins in the cytoplasm that are NB-LRR resistant genes are handled by P.infestans and therefore the genetic source for building resistance has to be generated. One way of doing it is to understand the cell surface receptors which can manage the extracellular danger signals and further can control the pathogen resistance. The receptors present in the membranes playing defensive roles are receptor like proteins called Cf4 and Ve1 which were found to impart resistance against fungi like Cladosporium fulvum and Verticillium dahliae respectively.
The pathogens are also recognized by receptors like kinases or RLKs. The RLKs that bear leucine-rich repeat domains like FLS2 and EFR were considered as disease resistance genes. Other RLK enzymes vital in plant defense are L-type lectin receptor kinases or LecRKs. The LecRK-1.9 receptor was found to bind with an RGD tripeptide motif of RXLR effector protein of P. infestans through its extracellular lectin domain. In another study, it was identified that Arabidopsis LecRK-1.9 mutant lines were susceptible to the P.brassicae. Over-expression of this protein in the plant lines developed resistance in the plants against P.brassicae.
The immunity in Arabidopsis was also found to be due to other proteins like LecRK-V.5 and LecRK-V1.2. In the present study, the disease development is reduced in potato as well as in Nicotiana benthamiana plants with the expression of LecRK-1.9. Therefore, transfer of LecRK-1.9 protein to other Solanaceous family members in the breeding methods will develop late blight resistance in these plants against P.infestans.
Results of this study
Transgenic potato lines, also called TPLs expressing Arabidopsis LecRK-1.9 were generated to impart resistance to potato plants against Phytophthora infestans. The potato cultivar 'Desiree' was transferred with the plasmid (pK-35S-LecRK-1.9) consisting of entire LecRK-1.9 coding sequence with the 35S-CaMV promoter through Agrobacterium mediated transfer. The expression levels of LecRK-1.9 in three transgenic plants TPL30, TPL36 and TPL42 were measured by Q-RT-PCR with the help of total RNA isolated from potato as a template. TPL30 plants showed lower expression while in TPL36 plants showed the highest expression of LecRK-1.9.
Impact of LecRK-1.9 expression on plant development in potato
The transgenic potato plants were transferred from culture medium into the soil and their growth was monitored in 6 weeks period. The leaves of the transgenic plants were deformed compared to those of Desiree plants. The changed phenotype severity differed among the three TPLs and the phenotype change was according to the LecRK-1.9 mRNA levels. The phenotype was severe in TPL30 having a low expression of LecRK-1.9, while wrinkles were more in TPL36 and TPL42 expressing higher levels of LecRK-1.9. The venation in TPL36 and TPL-42 was palmate type and that of in 'Desiree' was pinnate type. The tuber skin color of TPLs was yellow, while it was red color in recipient cv Desiree.
LecRK-1.9 increases late blight resistance in potato
Phytophthora infestans isolates of various origins were tested for their infecting capacity on TPL30, TPL36 and TPL42. All isolates of Phytophthora infestans could exhibit 100 percent of infection efficiency towards susceptible cultivar Desiree, while infection efficiency on TPLs was decreased significantly. The P.infestans isolate 88069 could show larger lesions on cultivar Desiree four days of post inoculation while it showed comparatively smaller lesions on three transgenic plants. The TPL30 transgenic plant showed less resistance towards P.infestans than the other two transgenic plants as the expression levels of Arabidopsis LecRK-1.9 were lower in TPL30. Therefore, constitutive expression of Arabidopsis lectin receptor kinase LecRK-1.9 in potato increased resistance in the plant against P.infestans.
Late blight resistance in Nicotiana benthamiana is increased by LecRK-1.9
The researchers in this study have expressed transgenically LecRK-1.9 tagged with GFP into Nicotiana benthamiana leaves by the method of agrofiltration. The level of expression was tested with Q-RT-PCR technique and the transcripts were measured sufficiently after 3 days of agrofiltration. Fluorescence protein was tagged to clearly identify the expressed LecRK-1.9 gene. The infection lesions on the agrofiltrated Nicotiana benthamiana leaves expressing LecRK-1.9-GFP were restricted to a large extent than on those leaves expressing gus. Large differences in lesion appearance was observed three days of post inoculation, while no significant differences were observed when agrofiltrated N.benthaminana plants were inoculated with Botrytis cinerea fungus.
So, the results altogether convey that expression of LecRK-1.9 increases resistance in the Solanaceous plants like Nicotiana benthamiana and potato against Phytophthora infestans.
Klaas Bouwmeester, Miao Han, Rosario Blanco-Portales, Wei Song, Rob Weide, Li-Yun Guo, Edwin A.G. van der Vossen and Francine Govers. The Arabidopsis lectin receptor kinase LecRK-1.9 enhances resistance to Phytophthora infestans in Solanaceous plants. Plant Biotechnology Journal (2014) 12, pp.10-16.
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