The Internal Glycine-Rich Motif and Cysteine Suppress Several Effects of the HpaG_Xooc Protein in Plants

Abstract

HpaG(Xooc), produced by Xanthomonas oryzae pv. oryzicola, is a member of harpin group of proteins that stimulate plant growth, hypersensitive cell death (HCD), and pathogen defense. The protein contains two copies of the glycine-rich motif (GRM), a characteristic of harpins, and a cysteine, which is absent in other harpins. Genetic modification generated the pro-tein mutants HpaG(Xooc)MG (MG) by deleting GRMs and HpaG(Xooc)C47T (C47T) by replacing cysteine with threonine. When applied to tobacco plants, C47T and MG were 1.2- and 1.7-fold stronger, respectively, than HpaG(Xooc) in inducing HCD, which occurred consistently with expression of the marker genes hin1 and hsr203. The proteins markedly alleviated infection of tobacco by Tobacco mosaic virus and Arabidopsis and tomato by Pseudomonas syringae. Treating tobacco plants with HpaG(Xooc), C47T, and MG decreased the viral infection by 58, 81, and 92%, respectively. In Arabidopsis and tomato plants treated with HpaG(Xooc), C47T, or MG, P. syringae multiplication was inhibited; bacterial population multiplied in 5 days in these plants were ca. 160-, 1,260-, or 15,860-fold smaller than that in control plants. So pathogen defense was induced in both plants. Defense-related genes Chia5, NPR1, and PR-1a were expressed consistently with resistance. In response to HpaG(Xooc), C47T, and MG, aerial parts and roots of tomato plants increased growth by 15 and 53%, 25 and 77%, and 46 and 106%, relative to controls. The expansin gene, EXP2, involved in the cell expansion and plant growth was expressed coordinately with plant growth promotion. These results suggest that the presence of GRM and cysteine in HpaG(Xooc) represses the effects of the protein in plants.

Nicotiana tabacumTTG1 contributes to ParA1-induced signalling and cell death in leaf trichomes

“…As shown in Fig. 7, both events were observed similarly in WT and RNAi plants responding to the chemical elicitors, salicylic acid (Ryals et al, 1996) and paraquat (Ge et al, 2007), in contrast to water, or responding to proteinaceous harpin elicitors, HrpN Ea (Peng et al, 2003;Wei et al, 1992), HpaG Xoo (Liu et al, 2006) and HpaG Xooc , in contrast to EVP. Upon treatment with each elicitor, chromatin condensation and cell death appeared 4-6 hours earlier in leaf trichomes than in mesophylls.…”

“…Similar signalling processes can be induced by different types of HCD elicitors, including harpin proteins of plant pathogenic bacteria Dong et al, 1999;Liu et al, 2006;Wei et al, 1992) and the chemical elicitors SA (Ryals et al, 1996) and paraquat (Ge et al, 2007). The proteinaceous and chemical elicitors induce chromatin condensation and cell death first in leaf trichomes and then in mesophylls in RNAi plants, as they do in WT.…”

“…Confirmed gene clones were subjected to prokaryotic transformation and expression Liang et al, 2007). The mutant C51S was obtained through sitedirected mutagenesis (Liu et al, 2006) conducted by PCR with the MutanBEST Kit (Takara Biotech, Dalian, China) and specific primers 5Ј-TCCGCGTCGA -CGGCGTCCAA-3Ј and 5Ј-CATGAGCTTGTACTGCTCCGTCGTGGG-3Ј. The GFP gene used to construct C51S-eGFP was cloned from the vector pEGFP-C1 (Clontech Laboratories, Palo Alto, CA).…”

“…After transformation and sequencing, recombinant E. coli BL21 cells were incubated to produce protein preparations . Proteins were quantified (Liu et al, 2006) and used at 10 μg/ml.…”

“…Primers were 5Ј-ATGGATAATTCAGCTCCAGATTC-3Ј and 5Ј-TCAAACTCTAAGGAGCTGCAT-3Ј. S94F was obtained through site-directed mutagenesis (Liu et al, 2006) conducted by PCR with the MutanBEST Kit (Takara Biotechnology, Dalian, China) and specific primers 5Ј-CGTCCTTCCTTCGGAGATCTC-3Ј and 5Ј-ACGGAGAGAAGGACT -GAAC-3Ј. The GFP gene used to construct ParA1-eGFP was cloned as described above.…”