The Functional Characterization Of A Laccase Gene GhLac1 In Cotton Broad-spectrum Resistance

Plants are constantly threatened by the aggression of a wide spectrum of pathogens and herbivores in the terrestrial phyllosphere,which cause massive yield and quality losses annually.Due to the never stopped co-evolution and mutual selection between plants and biotic invaders,plants have evolved a sophisticated and multilayered surveillance system to perceive and distinguish different invaders,and to launch prompt and effective defense responses.In the field conditions,plants are always simultaneously attacked by different pathogens and herbivores.The traditional breeding strategy always resulted in enhanced resistance to some invaders but increased susceptibility to others.The most economic and efficient approach is to enhance the plant?s broad-spectrum biotic stress resistance through genetic engineering.The plant cell wall,a plastic and intricate structure surrounding every plant cell,was regarded not only as a passive barrier against pathogens and pests but also a monitoring system to perceive the invaders and to induce the defense responses.Genes encoding enzymes involved in cell wall integrity may represent useful candidates for manipulating the plant defense responses.Therefore,a suppression subtractive hybridization library was constructed to identify genes expressed during cell wall regeneration in cultured protoplasts of cotton,from which we identified a cotton laccase gene GhLac1.The biofunctions of GhLac1 in cotton resistance to Verticillium dahilae,cotton bollworm and cotton aphid are as follows:1.The cloning and sequence analysis of GhLac1 in cottonWe isolated and identified three expressed sequence tags（GI:213047554,GI:213047555 and GI:213047556）that are homologous sequences of GaLAC1（Gossypium arboretum secretory laccase,GI:40218370）from the suppression subtractive hybridization library of cotton cultured protoplasts during cell wall regeneration.And we cloned the full length of GI:213047556 which showed the most significant induced expression pattern by V.dahliae,JA and H₂O₂ treatments among the three ESTs and named it GhLac1.Gh Lac1 contains the predicted copper binding domain conserved among plant laccase proteins and showed the highest identity with the laccase protein from Theobroma cacao（GI:590604253）.Quantitative real-time PCR（qRT-PCR）analysis confirmed that GhLac1 was expressed in various organs and predominantly expressed in roots.The induced expression pattern showed GhLac1 was strongly induced by V.dahliae,JA and H₂O₂ treatments but showed no response to SA,which implied that GhLac1 might participate in cotton defense aganist V.dahlia and involved in JA/H₂O₂ mediated signalling pathway.2.Overexpressing GhLac1 in cotton promoted lignin synthesis and resulted in enhanced resistance to Verticillium dahilae,cotton bollworm and cotton aphidTo investigate the biofunction of GhLac1 in cotton,the over-expression（35S::GhLac1）and RNA interference（35S::RNAi GhLac1）constructs that under the control of 35 S promotor were generated and transformed into cotton.The over-expression lines of GhLac1 were subjected to V.dahilae strain V991,cotton bollworm and cotton aphid.Compared with wild type,the over-expression lines showed enhanced resistance to V991,cotton bollworm and cotton aphid with increased lignin content in roots,stem and young leaves.3.Down-regulating GhLac1 in cotton resulted in accumulated flavonoids and JA and enhanced plant resistance to Verticillium dahilae and cotton bollwormCompared with wild type,the lignin deposition in roots,stem and young leaves were significantly decreased in the RNAi lines of GhLac1.Whereas,the expression level and enzyme activity of phenylalanine ammonia lyase（PAL,EC.4.3.1.5）was increased in the RNAi lines than that of wild type,the results implied that down-regulating GhLac1 might lead to a redirection of the metabolic flux in the phenylpropanoid pathway to the branch of flavonoids synthesis.Further experiments confirmed that the total flavonoids and gossypol equivalents contents were significantly increased in the roots and young leaves of RNAi lines,and those secondary metabolites showed obviously toxic effects on the growth of V.dahilae mycelia and cotton bollworm.Moreover,we also found that the JA and JA-Ile contents also showed significant accumulation in the RNAi lines of GhLac1,which resulted in constitutively activated JA mediated signalling pathway and enhanced plant resistance to V.dahilae and cotton bollworm.Thus,the accumulated secondary metabolites and activated JA mediated signalling pathway jointly contributed to the enhanced resistance to V.dahilae and cotton bollworm in the RNAi lines of GhLac1.4.Down-regulating GhLac1 in cotton led to the susceptibility of cotton to aphidThe RNAi lines of GhLac1 were subjected to cotton aphid,compared with wild type,the RNAi lines showed enhanced susceptibility to cotton aphid.On the one hand,the reduced lignin content in the feeding site of the RNAi lines of GhLac1 might facilitate the insertion of aphid stylets;on the other hand,the constitutively activated JA signalling pathway suppressed the SA signalling pathway in RNAi lines.According to previous reports,the plants resistance to sap-sucking herbivores like aphid mainly depended on the SA mediated signalling pathway.Thus,the reduced lignin content and the observed reduced activity of the SA signalling pathway accounted for the susceptibility to cotton aphid in the RNAi lines.5.The altered cell wall as a stress generator affected jasmonates levels in RNAi lines of GhLac1According to previous studies,the increased cell wall pectic polysaccharides in apoplast could mimic the cell wall damage from the penetration by fungal pathogens and chewing by herbivores.The cell wall pectic polysaccharides could be perceived by a diverse set of plasma membrane-localized sensors and subsequently to initiate the downstream immune responses.In the lignin synthesis deficient plants,the altered cell wall integrity due to decreased lignin content also showed increased pectic polysaccharides content in the apoplast and the activated defense responses.To investigate the possible reason（s）for the accumulated JA and JA-Ile content in RNAi lines of GhLac1,we detected the cell wall pectic polysaccharides content in wild type and GhLac1 transgenic lines.The results showed that the RNAi lines released much more cold water-soluble and EDTA soluble pectic materials than did WT,and the cold water-soluble pectic material could induce the JA content in cotton suspension cultured cells.Thus,the accumulated JA and JA-Ile content might be induced by the increased cell wall pectic materials in the RNAi lines.