Bioelicitor And Redox Signaling Components Differentially Regulate Plant Growth And Disease Resistance

Due to public concerns on pesticide use in crops, exploration on alternative methods has been a global effort to secure crop. Biocontrol bacteria and natural products from various sources show a great potential of agricultural use in disease control and crop product improvement. Pseudomonas and Bacillus species is an important class of bioeontrol bacteria; and they protect plants with many mechanisms. Moreover, biocontrol bacteria can promote seed germination and plant growth with indirectly increasing disease resistance. Plant responses are induced by the biocontrol bacteria colonization on plant roots and play a role in disease control depending on the response speed and magnitude in contrast to the infection to plants by pathogens. Natural products which have a potential in crop improvement are various in nature and sources. HpaG_Xoo, a harpin produced by Xanthomonas oryzae pv. oryzae, stimulates plant growth and resistance against pathogens and insects. Moreover, the biocontrol bacteria colonization on plant roots activates induced systemic resistance(ISR), which antagonizes SAR and provides plants with a distinct battery of defense arsenal. Therefore, a combinative use of biocontrol bacteria and the type-Ⅲeffectors could be more effective than use of either of them in crop improvement. Previously, we used P. cepacia P6854 and B. subtilis B-916 to control rice sheath blight with desired results. We have introduced HpaG_Xoo into rice(variety R109), generating several transgenic lines. They were improved in disease resistance. Here we show that the HpaG_Xoo-expressing rice line 1(HER1) increases growth and activates defenses toward salinity and pathogens. We present evidence that P. cepacia P6854 and B. subtilis differentially affects growth and disease resistance in R109 and HER1.Elicitins are a peculiar of proteins produced by oomycetes in the genera Phytophthora and Pythium, and ParA1 is 10 kDa elicitor produced by P. parasitica var. nicotinanae, host special elicitor, sensitive to heat and protease K in vitro. Elicitins induce not only plant hypersensitive response(HR) but also defense reponses. In order to get more effective and more economical recombinant bacteria than the recombinant E. coli BL21(DE3) that expressing ParA1 and HpaG_Xoo singly, we constructed a bacterium expressing HpaG_Xoo and ParA1 simultaneously. The protein of the bielicitor bacterium has been tested for its effect on tobacco micro-HR and the resistance against tobacco mosaic virus.Riboflavin is an important ingredient of flavin mononucleotide(FMN) and flavin adenine dinucleotide(FAD) in bacteria and eukaryotic organisms. FMN and FAD participate in many enzyme catalizing reactions, so riboflavin is a multifunctional vitamin which is essential for the maintenance of life. The two enzymes, lumazine synthase and riboflavin synthase, catalyze the last two steps in the biosynthesis of riboflavin. The two enzymes represent attractive targets for the development of drugs against bacterial pathogens, because the inhibitors of these enzymes are not likely to interfere with the enzymes of the mammalian metabolism. The genes of many bacteria and yeasts have been cloned, but there are only little reports on plants and no research on rice. It has nearly been reported that the cos1 mutation(LS mutation) restores the coil-related phenotypes, including defects in JA sensitivity, senescence and plant defense responses in Arabidopsis, but there have no research about the functions on growth, disease resistance, active oxygen and hypersensitive cell death of the two genes of rice by so far. It is worth to research.Thioredoxins are small proteins catalyzing thiol-disulfide interchange and are involved in the regulation of the redox environment of the cells. The plant thioredoxin system is particularly complex since many thioredoxin isoforms are found in plants and they are multifunctional proteins. Based upon primary sequence analysis and subcellular localization, thioredoxins can be classified into different groups and subgroups. The functions of tobacco thioredoxin h-like protein have not been reported, we try to research this gene actions on plant growth and disease resistance.This study is focused on crosstalk between representative and distinct agencies that induce plant defense and growth, and functions of pivotal regulators in redox-based signal transduction that regulates both effects in plants. The interactions of HpaG_Xoo with P. cepacia or ParA1, and functions of the genes encoding lumazine synthase and riboflavin synthase from rice, and a gene encoding h-like thioredoxin from tobacco, as well, have been studied by multiple methods.1 Bioelicitor and PGPR interact to affect plant growth and disease resistance Expression of HpaG_Xoo, a bacterial type-Ⅲeffector, in transgenic plants induces disease resistance. Resistance also can be elicited by biocontrol bacteria. In both cases, plant growth is often promoted. Here we address whether biocontrol bacteria and HpaG_Xoo can act together to provide better results in crop improvement. We studied effects of P. cepacia and B. subtilis on the rice variety R109 and the hpaG_Xoo-expressing rice line HER1. Compared to R109, HER1 increased growth, grain yield and defense responses toward diseases and salinity stress. Colonization of roots by P. cepacia or B. subtilis caused some increase, in contrast to controls, in root growth of R109. Growth of R109 leaves and stems and HER1 roots were effected a little but leaves and stems of HER1 was inhibited. When P. cepacia and B. subtilis colonization was subsequent to plant inoculation with Rhizoctonia solani, a pathogen that causes sheath blight, the disease was less severe than controls in both R109 and HER1; HER1, nevertheless, was more resistant, suggesting that P. cepacia or B. subtilis and HpaG_Xoo cooperate in inducing disease resistance. Several genes that critically regulate growth and defense behaved differentially in HER1 and R109 while responding to P. cepacia or B. subtilis. In R109 roots, the OsARF1 gene, which regulates plant growth, was expressed in consistence with growth promotion by P. cepacia or B. subtilis. Inversely, OsARF1 expression was coincident with inhibition in growth of HER1 leaves. In both plants, the expression of OsEXP1, which encodes an expansin protein involved in plant growth, was concomitant with growth in leaves and roots, in response to P. cepacia or B. subtilis. We also studied OsMAPK, a gene that encodes a mitogen-activated protein kinase and controls defense responses toward salinity and infection by pathogens in rice. In response to P. cepacia or B. subtilis., an early expression of OsMAPK was coincident with R109 resistance to the disease, while HER1 expressed the gene similarly whether P. cepacia was present or not. Evidently, P. cepacia or B. subtilis and HER1 interact differently in rice growth and resistance. Whereas combinative effects of P. cepacia or B. subtilis and HpaG_Xoo in disease resistance have a great potential in agricultural use, it is interesting to study mechanisms that underlie interactions involving biocontrol bacteria, type-Ⅲeffectors and pathogens.HpaG_Xoo and ParA1 are two important elicitors produced by plant pathogens, the genes parA1 and hpaG_Xoo were cloned by polymerase chain reaction and ligased into the expressing vector pET30a(+), transformed into E. coli BL21(DE3). The protein of BL21:: parA1:: hpaG_Xoo (ParA1:: HpaG_Xoo) was extracted, and electrophoresed by Tris-Tricine SDS-PAGE, 15 kDa and 10 kDa two proteins were got. The ParA1 and HpaG_Xoo were identified expressing synchronously because of different macroscopic hypersensitive response(HR) of tobacco leaves injected with the proteins boiled 30 min or not. ParA1:: HpaG_Xoo induced stronger microscopic hypersensitive response(micro-HR), systemic acquired resistance(SAR) to tobacco mosaic virus and expression of pathogen related genes, PR1a and PR1b than ParA1 or HpaG_Xoo only did.2 The roles of lumazine synthase and riboflavin synthase in plant growth and defenseBased on transgenic and molecular studies, the functions of both enzymes from rice were researched in this study.OsLS and OsRS are first cloned. OsLS have 666 nucleotides. The nucleotide sequence identities of LS genes of Oryza sativa, Arabidopsis thaliana, Spinacia oleracea and Nicotiana tabacum are between 68-76％, and the genes are in the same branch of phylogenetic tree. The nucleotide sequences of LS genes of Magnaporthe grisea and Photobacterium phosphoreum have no identity, but the amid acid sequences of them have 33％and 48％identities and the genes are in different branches of phylogenetic tree. OsLS protein with His-tag(29.85 kDa) was expressed in E. coli. OsLS protein has 221 aa, its molecular weight is 22, 471.44 Da, and pI is 10.01.100μg/ml OsLS protein aqueous solution cann't induce tobacco hypersensitive response, meaning this protein is not bad for plants. The protein, analyzed online, is localized in chloroplast, as the same as the location of the LS proteins of A. thaliana, spinach and tobacco. Its tertiary structure has 4μfolds, surrounded by 5αhelixes. OsRS has 1662 nucleotides, having 100％identity with Gene ID OJ1111H02. Predgene04. NCBI Blast analyed that OsRS has no identity with the RS sequences of A. thaliana, Candida albicans, C. famata, Filobasidiella neoformans, Sinorhizobium meliloti, Bartonella elizabethae, Saccharomyces cerevisiae, Pichia guilliermondii, P. phosphoreum, Schizosaccharomyces pombe and E. coli, and the genes are in different branches of phylogenetic tree. OsRS protein with His-tag was expressed in E. coli, whose molecular weight is 66.37 kDa. 100μg/ml OsRS protein aqueous solution can't induce tobacco hypersensitive response, meaning this protein is not bad for plant. It has 553 aa, the molecular weight is 59, 589.80 Da and pI is 6.00. The protein analyzed online is localized in chloroplast, and its tertiary structure has 5βfolds, surrounded by 6αhelixes.OsLS and OsRS trangenic tobacco have higher levels of free riboflavin, FMN and FAD than that of the blank vector transgenic tobacco(VECT), and the OsLS transgenic tobacco (LST) have higher level than OsRS transgenic ones(RST). LST and RST have better growth in infancy and youthful times than the wild type(WT) and VECT. Expansin genes, which enloose cell wall and enhance plant growth, of LST and RST have higher expression levels than VECT and WT. RST growed better than LST. OsLS and OsRS have no effect on programmed cell death in transgenic tobacco lines compared with WT and VECT, the marker genes hin1 and hsr203 have similar expression levels in them. The disease resistances of LST and RST were enhanced, the pathogenesis-related genes(PR) were induced stronger than control(CK), and LST have better resistance than RST. Ethylene is an important signal molecular related to development and disease resistance. LST and RST can produce more ethylene than CK, this may resulted into fast growth and strong disease resistance. Active oxygen is important for plant disease resistance, and the active oxygen of high level destroy plant disease resistance. Without TMV inoculation, LST and RST have no difference with CK, but LST and RST were haunted by less active oxygen stress, and especially LST has the least one, this means OsLS has sronger energy to eliminate active oxygen stress than OsRS.Rice immature embryo calli were transformed with the overexpressing vector and hairpin unit of OsLS and OsRS by soaking the plant material with an appropriate Agrobacterria tumefaciens EHA105 suspension. OsLS and OsRS overexpressing rice(LSR and RSR) and OaRS partly silencing lines(SiRS) have been got, but OsLS haipin transformed rice(SiLS) was not got. The rice calli transformed by OsLS hairpin rice couldn't differentiate. The calli transformed by OsLS overexpressing vector rice grew and differentiated slower than those transformed by the blank vector, but the rice calli transformed by OsRS overexpressing vector grew and differentiated fast. The rice calli transformed by OsRS hairpin vector grew and differentiated slower than CK. Four-month old LSR and RSR have higher levels of free riboflavin, FAD and FMN than CK. SiRS have lower levels of riboflavin, FAD and FMN than CK. RSR have more tillers than CK, but LSR and SiRS have fewer tillers than CK. OsMOC1, which controls tiller formation and OsGRF1, which expresses in actively growing parts, have higher expression levels in RSR than CK, but have lower expression in LSR and SiRS than CK. All lines have no marked difference in programmed cell death. The disease resistance of LSR was enhanced the most among RSR, VECR, SiRS and LSR. RSR were enhanced a little disease resistance than VECR. The defenee response of SiRS were damaged absolutely. The expression levels of pathogenesis-related genes, OsPR1b and OsPR10, were in the same trend with rice blight resistance. LSR and RSR were effected by less active oxygen stress than VECR, but SiRS haunted by more active oxygen stress than CK. So OsLS and OsRS are very important, OsLS induces disease resistance and inhibits growth, but OsRS induces growth and disease resistance, they all eliminate active oxygen stress.3 Tobacco thioredoxin signaling inhibits plant growth but induces disease resistance and does not affect programmed cell deathNicotiana tabacum thioredoxin h-like protein complete CDS sequence was cloned, with the conserved active domain WCGPC. The amino acid sequence identities of TRX with those of N. tabacum(NtTRX-hl, AF435818), Triticum aestivum(Tal, AF438359), Hordeum vulgare(Hvhl, AF435815) and Arabidopsis thaliana(Ath-tl, AAG51342) are 71％, 72％and 78％respectively, and the genes are in the same branch of phylogenetic tree. The identities with those of Lycopersicon esculentum(LeCITRX-pt, AF261142), Solanum tuberosum(StCDSP32, Y09987) and N. alata(Nah, DQ021448) are only 30％, 24％and 37％respectively, and the genes are localized in the different branches of phylogenetic tree. TRX protein was expressed with His-tag(5, 426 Da) in vitro, the molecular weight is 22, 260 Da. 150μg/ml TRX protein couldn't induce tobacco hypersensitive response, meaning the protein is not bad to plant. TRX protein has 152 aa, its molecular weight is 17, 022.20 Da, and its pI is 4.53. This protein is most possible in mitochondrial matrix space, and may be in cytoplasm, microbody(peroxisome) or mitochondrial inner membrane. TRX tertiary structure has 2α-helixes and 5β-folds.TRX partially silenced tobacco plants(SiTRX) grew faster than the control(CK), the difference is marked, and the expansin genes, which enloose cell wall and enhance plant growth, NtEXP1, NtEXP2 and NtEXP6 have higher expression levels than CK. TRX overexpressing plants(OvTRX) have the reverse results compared with SiTRX. TRX silence and overexpression have no effect on hypersensitive cell death compared with CK, the marker genes hin1 and hsr203 had similar expression levels in LST, RST and CK. The disease resistance of SiTRX was damaged, the pathogenesis-related genes PR1a and PR1b were induced less than CK, and this means TRX disturbs these genes expression, in the other side OvTRX have better TMV resistance and higher pathogenesis-related gene expression levels than CK. Without TMV inoculation and within 48 inoculation, active oxygen levels were similar in SiTRX, OvTRX and CK, but 48 hours postinoculation SiTRX produced more active oxygen than CK, but OvTRX produce less than CK. Clearly, the TRX gene inhibits plant growth but induces disease resistance and doesn't affect hypersensitive cell death. ConclusionIn a conclusion, we have some results about the interaction of HpaG_Xoo with P. cepacia, B. subtilis or ParA1, the functions of lumazine synthase and riboflavin synthase from rice and thioredoxin from tobacco. First, there are some interactions between HpaG_Xoo and P. cepacia, B. subtilis. or ParA1, which effected on plant disease resistance and hypersensitive response. Second, OsLS and OsRS have important roles on plant growth and disease resistance. OsRS enhances growth, OsLS strongly induces disease resistance, and the two genes doesn't affect programmed cell death. The two genes have important potentials on the culture of the varieties of short-time cycle, high yield and strong disease resistance. Finally, TRX inhibits tobacco growth, induces disease resistance and doesn't affect hypersensitive cell death.The theory bases and transgenic plants are prepared to explore better plant protection strategy, and to culture the varities with better growth and disease resistance than CK, to study the roles of riboflavin pathway and redox signals in plant disease resistance and growth, and to look for their target proteins.