Sphingolipid Metabolism In Relation To Insect Resistance In Plants

In plants,Sphingolipids and their metabolic precursors,the long-chain bases(LCBs)act as bioactive molecules in the immune response.LCBs play essential roles in many aspects of plant development including cell growth,differentiation,proliferation,cell death as well as responses to different stresses.Biosynthesis of these LCBs involves the condensation between L-serine and palmitoyl-CoA in a reaction catalyzed by the enzyme serine palmitoyltransferase(SPT).To elucidate the function of SPT as well as sphingolipids as insect resistance the present study was conducted.We focused on a gene named OsLCB2?l cloned from rice.OsLCB2?l open reading frame containing 1470 bp,encoding 489 amino acids polypeptide with 90,84,and 82%identity to Zea mays SPT2,Nicotiana banthamiana SPT and Arabidoposis thaliana.LCB2 respectively.The gene was induced by brown planthopper(BPH)feeding.OsLCB2?1 gene expression was high in resistant variety like Mudgo and IR64,it was negatively correlated with BPH infestation reaction score.mRNA level of this gene in varieties Taiping and IR64 showed significantly higher when plant infest with 20 BPH nymph/seedling.Gene expression was observed in 1,2,4,8,16 and 24 hrs after infestation of BPH nymph in the same 3 varieties.Expression was high at 2 hrs of infestation in IR64 and Taiping.Among different plant parts of above three rice varieties gene expression found significantly higher in leaf blade of IR64 and leaf sheath of Taiping.Gene expression was comparatively lower in root compared to leaf blade and leaf sheath in the variety IR64 and Taiping.But in TN1 gene expression showed significantly lower in all plant parts.Transcript levels of OsLCB2?1 gene was also compared in the seedling,vegetative and reproductive stages of rice plant of same three rice varieties.Significantly higher gene expression was observed in maximum tillering stage of IR64 and Taiping.OsLCB2?1 gene was transformed to Arabidopsis WT and mutant plants through agrobacterium floral deep transformation.The mutants show reduced stature with smaller and narrower leaves than wild-type plants.Transformation of OsLCB2?1 into the mutant plant showed that the OsLCB1?1 can complement the phenotypes of mutant.Overexpressed plant showed similar phenotype to wild type.The T-DNA border sequence and insertion position of transgenic plants were confirmed by TAIL PCR.The insertion site located upstream 1 kb of AT5G56900 and downstream 2.7 kb of AT5G56890,without any annotated genes broken for the over-expressed plant.The insertion site located inside of AT5G24630 for the mutant complementation plant.Quantitative Real Time PCR in 4-week-old leaves of Arabidopsis plant revealed that the gene expression was increased about 33 and 28 fold in over-expressed and mutant complementation plant respectively.Sphingolipid measurement profiling showed that over-expressed plants have high LCBs and ceramidases.Trihydroxylated LCB phytosphingosine(t18:0),which is more abundant,increased 1.7 fold and phytoceramide increased 1.3 fold in over-expressed plant compared to wild type.Over expression of OsLCB2?1 gene showed resistance to insect.Population size of green peach aphids(Myzus persicae)in over-expressed plants was smaller than that in mutant and wild type plant after 14 days.In mutant the population of aphid was found almost double suggesting that overexpression of OsLCB2?1 might improve aphid tolerance of the transgenic plants due to increase of sphingolipid level.To elucidate whether aphid feeding behavior was affected by overexpression of OsLCB2?1 gene we compared electrical penetration graph(EPG)recordings of M.persicae on wild type mutant,overexpressed and mutant complementation plants.The time to the first probe was similar in all plant types.Compared to wild type M.persicae on overexpressed plant needed 1.6 times more time to the first phloem phase.Aphids on overexpressed plant spent significantly less time and took smaller number of time salivating into the phloem,ingesting phloem sap and sustained phloem than aphids on mutant and wild type.Moreover,aphid showed a significantly longer duration of the non-probing phase on overexpressed than on WT,mutant complementation and mutant plants.Waveform F,associated with derailed stylet penetration,was also observed for a significantly longer time and in a larger number on overexpressed arabidopsis plants.On the other hand,t he aphids spent less time taking up xylem sap from over-expressed plant as was indicated by a shorter time and smaller number of waveform G.Our study revealed that OsLC2?1 influences FB1-induced cell death.