Inhibition of conidial growth of Venturia inaequalis by the extracellular protein fraction from the antagonistic bacterium Pseudomonas fluorescens Bk3

The present study investigated the inhibitory effect on the conidial germination of Venturia inaequalis by using living whole cells, extracellular protein fraction and individual proteins from the non-pathogenic antagonistic bacterium Pseudomonas fluorescens Bk3.The bacterial suspension of P. fluorescens Bk3 from growth in minimal medium showed up to 73% inhibition of conidial germination after 7 days of pre-incubation. Furthermore, this inhibitory effect could also be shown by the extra cellular protein fraction of P. fluorescens Bk3. The protein solution obtained from liquid culture in LB medium showed 100% inhibition of conidial germination after 1 day of pre-incubation. Since the solution contained at least 10 major proteins these proteins were extracted from the gel and subsequently re-natured for functional studies. After re-naturation individual proteins were applied on the conidia of V. inaequalis to see their impact on the conidial germination. Out of these 10 proteins three showed inhibitory effects (20–42%). De novo sequencing of these three proteins were carried out by ESI Q-ToF mass spectrometry and they were identified as an extracellular solute-binding protein, an extracellular alkaline metalloprotease and a peptidoglycan-associated lipoprotein. The proteolytic activity of the metalloprotease could also be confirmed with activity staining using casein as a substrate.     

Biological control of apple blue mold with Pseudomonas fluorescens

Pseudomonas fluorescens isolate 1100-6 was evaluated as a potential biological control agent for apple blue mold caused by Penicillium expansum or Penicillium solitum. Both the wild-type isolate 1100-6 and a genetically modified derivative labeled with the gene encoding the green fluorescent protein (GFP) were compared. The P. fluorescens isolates with or without GFP equally reduced the growth of Penicillium spp. and produced large zones of inhibition in dual culture plate assays. Cell-free metabolites produced by the bacterial antagonists reduced the colony area of Penicillium isolates by 17.3% to 78.5%. The effect of iron chelate on the antagonistic potential of P. fluorescens was also studied. The use of iron chelate did not have a major effect on the antagonistic activity of P. fluorescens. With or without GFP, P. fluorescens significantly reduced the severity and incidence of apple decay by 2 P. expansum isolates after 11 d at 20 degrees C and by P. expansum and P. solitum after 25 d at 5 degrees C when the biocontrol agents were applied in wounds 24 or 48 h before challenging with Penicillium spp. Populations of P. fluorescens labeled with the GFP were determined 1, 9, 14, and 20 d after inoculation at 5 degrees C. The log CFU/mL per wound increased from 6.95 at the time of inoculation to 9.12 CFU/mL (P < 0.05) 25 d after inoculation at 5 degrees C. The GFP strain did not appear to penetrate deeply into wounds based on digital photographs taken with an inverted fluorescence microscope. These results indicate that P. fluorescens isolate 1100-6 could be an important new biological control for apple blue mold.     

Erwinia amylovora modifies phenolic profiles of susceptible and resistant apple through its type III secretion system

Fire blight is a disease affecting Maloideae caused by the necrogenic bacterium Erwinia amylovora , which requires the type III protein secretion system (TTSS) for pathogenicity. Profiles of methanol-extractable leaf phenolics of two apple ( Malus  ×  domestica ) genotypes with contrasting susceptibility to this disease were analyzed by HPLC after infection. Some qualitative differences were recorded between the constitutive compositions of the two genotypes but in both of them dihydrochalcones accounted for more than 90% of total phenolics. Principal component analysis separated leaves inoculated with a virulent wild-type strain from those inoculated with a non-pathogenic TTSS-defective mutant or with water. The changes in levels of the various groups of phenolics in response to the virulent bacterium were similar between the two genotypes, with a significant decrease of dihydrochalcones and a significant increase of hydroxycinnamate derivatives. Differences between genotypes were, however, recorded in amplitude and kinetic of variation in these groups. Occurrence of oxidation and polymerization reactions is proposed, based on the browning process of infected tissues, but whether some by-products act in defense as toxic compounds remain to be tested. Among direct antibacterial constitutive compounds present in apple leaves, the dihydrochalcone phloretin only was found at levels close to lethal concentrations in both genotypes. However, E.   amylovora exhibited the ability to stabilize this compound at sublethal levels even in the resistant apple, rejecting the hypothesis of its involvement in the resistance of this genotype.     

Role of proteins in resistance mechanism of Pseudomonas fluorescens against heavy metal induced stress with proteomics approach

The genus Pseudomonas is a group of gram-negative, motile, rod-shaped bacteria known for their metabolic versatility. One of the species is Pseudomonas fluorescens, which has an efficient system for detoxification of industrial waste. Other aspects include, catabolic versatility, excellent root colonizing abilities and capacity to produce a wide range of antifungal metabolites. They are also known for their resistance and survival in the presence of several organic and inorganic pollutants. P. fluorescens has also been isolated from metal polluted water and soils but the elucidation of proteins responsible for its survival is still not clear. The aim of the study was to elucidate the differential protein expression of this bacterium when exposed to heavy metal stress, using two-dimensional electrophoresis. The proteins spo VG and enolase showed upregulation during the bacterial exposure to lead and copper. Hypothetical protein showed downregulation when bacterium was exposed to cobalt. Some proteins like xylosyltransferase, ORF 18 phage phi KZ, OMP H1 and translational elongation factor EF-Tu appeared only during their exposure to cobalt. These were absent in the control condition. Analysis of the differentially expressed proteins as well as the newly synthesized proteins along with the results obtained growth and enzyme activity indicate the involvement of all these factors in the survival of this organism in the presence of heavy metals.     

Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted protein

We report the identification of an ATP-binding cassette (ABC) transporter and an associated large cell-surface protein that are required for biofilm formation by Pseudomonas fluorescens WCS365. The genes coding for these proteins are designated lap for large adhesion protein. The LapA protein, with a predicted molecular weight of approximately 900 kDa, is found to be loosely associated with the cell surface and present in the culture supernatant. The LapB, LapC and LapE proteins are predicted to be the cytoplasmic membrane-localized ATPase, membrane fusion protein and outer membrane protein component, respectively, of an ABC transporter. Consistent with this prediction, LapE, like other members of this family, is localized to the outer membrane. We propose that the lapEBC-encoded ABC transporter participates in the secretion of LapA, as strains with mutations in the lapEBC genes do not have detectable LapA associated with the cell surface or in the supernatant. The lap genes are conserved among environmental pseudomonads such as P. putida KT2440, P. fluorescens PfO1 and P. fluorescens WCS365, but are absent from pathogenic pseudomonads such as P. aeruginosa and P. syringae. The wild-type strain of P. fluorescens WCS365 and its lap mutant derivatives were assessed for their biofilm forming ability in static and flow systems. The lap mutant strains are impaired in an early step in biofilm formation and are unable to develop the mature biofilm structure seen for the wild-type bacterium. Time-lapse microscopy studies determined that the lap mutants are unable to progress from reversible (or transient) attachment to the irreversible attachment stage of biofilm development. The lap mutants were also found to be defective in attachment to quartz sand, an abiotic surface these organisms likely encounter in the environment.     

Cloning and characterization of the Pseudomonas fluorescens ATP-binding cassette exporter, HasDEF, for the heme acquisition protein HasA

Two ATP-binding cassette (ABC) exporters are present in Pseudomonas fluorescens no. 33; one is the recently reported AprDEF system and the other is HasDEF, which exports a heme acquisition protein, HasA. The hasDEF genes were cloned by DNA hybridization with a DNA probe coding for the LipB protein, one of the components of the Serratia marcescens ABC exporter Lip system. P. fluorescens HasA showed sequence identity of 40 to 49% with HasA proteins from Pseudomonas aeruginosa and Serratia marcescens. The P. fluorescens Has exporter secreted HasA proteins from P. fluorescens and P. aeruginosa but not S. marcescens HasA in Escherichia coli, whereas the Has exporter from S. marcescens allowed secretion of all three HasA proteins. The P. fluorescens HasDEF system also promoted the secretion of the lipase and alkaline protease of P. fluorescens. Hybrid exporter analysis demonstrated that the HasD proteins, which are ABC proteins, are involved in the discrimination of export substrates. Chimeric HasA proteins containing both P. fluorescens and S. marcescens sequences were produced and tested for secretion through the Has exporters. The C-terminal region of HasA was shown to be involved in the secretion specificity of the P. fluorescens Has exporter.     

Biological Control of Groundnut Late Leaf Spot and Rust by Seed and Foliar Applications of a Powder Formulation of Pseudomonas fluorescens

Glasshouse and field trials were conducted to determine the efficacy of a talc-based powder formulation of Pseudomonas fluorescens strain Pf1 in controlling groundnut leaf spot ( Cercosporidium personatum ) and rust ( Puccinia arachidis ). Seed treatment with the talc-based powder formulation of the bacterium alone effectively reduced the severity of leaf spot and rust. When the treated seeds were sown in soil, the antagonist moved to the rhizosphere and multiplied well in it. Foliar application with the powder formulation effectively controlled the groundnut leaf spot and rust. P. fluorescens multiplied well in the phyllosphere after foliar application of powder formulation. Combined application of the P. fluorescens formulation to seed and foliage effectively controlled leaf spot and rust, and increased the pod yield in greenhouse and field tests.     

Malus hupehensis NPR1 induces pathogenesis-related protein gene expression in transgenic tobacco

Most commercially grown apple cultivars are susceptible to fungal diseases. Malus hupehensis has high resistance to many diseases affecting apple cultivars. Understanding innate defence mechanisms would help to develop disease-resistant apple crops. Non-expressor of pathogenesis-related genes 1 (NPR1) plays a key role in regulating salicylic acid (SA)-mediated systemic acquired resistance (SAR). MhNPR1 cDNA, corresponding to genomic DNA and its 5' flanking sequences, was isolated from M. hupehensis. Sequence analysis showed that the regulatory mechanism for oligomer-monomer transition of the MhNPR1 protein in apple might be similar to that of GmNPR1 in soybean, but different from that of AtNPR1 in Arabidopsis. No significant differences in MhNPR1 expression were found in M. hupehensis after infection with Botryosphaeria berengeriana, showing that MhNPR1 might be regulated by pathogens at the protein level, as described for Arabidopsis and grapevine. SA treatment significantly induced MhNPR1 expression in leaves, stems and roots, while methyl jasmonate (MeJA) treatment induced MhNPR1 expression in roots, but not in leaves or stems. The expression of MhNPR1 was highly increased in roots, moderately in leaves, and did not change in stems after treatment with 1-aminocyclopropane-1-carboxylic acid (ACC). SAR marker genes (MhPR1 and MhPR5) were induced by SA, MeJA and ACC in leaves, stems and roots. Overexpression of MhNPR1 significantly induced the expression of pathogenesis-related genes (NtPR1, NtPR3 and NtPR5) in transgenic tobacco plants and resistance to the fungus Botrytis cinerea, suggesting that MhNPR1 orthologues are a component of the SA defence signalling pathway and SAR is induced in M. hupehensis.     

Comparative adherence to human A549 cells, plant fibronectin-like protein, and polystyrene surfaces of four Pseudomonas fluorescens strains from different ecological origin

The main objective of this study was to compare the adherence properties of four Pseudomonas fluorescens isolates from different ecological niches (human tissue, rhizosphere, drinking water, and cow milk). The substrates used to test P. fluorescens adherence were as follows: cultured human respiratory epithelial cells A549, immobilized plant fibronectin-like protein, and polystyrene. For all the experiments, bacteria were grown at 27 degrees C. The adherence assay to human cells was performed at 37 degrees C, whereas adherence to fibronectin and polystyrene was done at 27 degrees C. The four strains tested adhered to A549 cells but showed different adherence patterns. At 3 h, the milk isolate showed an aggregative adherence phenotype, whereas the three other isolates showed a diffuse adherence pattern. With a longer incubation time of 24 h, the aggregative pattern of the milk isolate disappeared, the adherence of the clinical strain increased, the adherence of the water isolate decreased, and morphological changes in A549 cells were observed with the clinical, water, and soil isolates. The four strains tested formed biofilms on polystyrene dishes. The clinical and milk isolates were the more efficient colonizers of polystyrene surfaces and also the more adherent to immobilized plant fibronectin-like protein. There was no relation between bacterial surface hydrophobicity and P. fluorescens adherence to the substrates tested. The main conclusions of these results are that P. fluorescens is an adherent bacterium, that no clear correlation exists between adherence and ecological habitat, and that P. fluorescens can adhere well to substrates not present in its natural environment.     

不同抗性苹果品种应答轮纹病菌胁迫的差异蛋白质组分析

为鉴定不同抗性苹果(Malus domestica)品种响应轮纹病菌胁迫的抗性相关蛋白表达差异, 以抗病品种华月及易感品种金冠为试材, 采用高通量同位素标记定量(IBT)技术结合液相色谱-串联质谱(LC-MS)鉴定技术, 对病原菌处理前后抗、感病品种叶片的蛋白质组差异表达进行分析, 共鉴定出171个差异表达蛋白(DEPs)。GO富集及KEGG通路分析表明, 在细胞组分、分子功能和生物过程3类中共注释到686个GO条目, 其中52个DEPs注释于KEGG通路的18个显著差异途径(P<0.05)。亚细胞定位预测分析表明, 171个DEPs中有170个分别定位于8类细胞器。蛋白功能注释分析表明, 46个DEPs注释于7类抗性相关蛋白, 包括类甜蛋白、过氧化物酶、多酚氧化酶、过敏原蛋白、几丁质酶、内切葡聚糖酶以及主乳胶蛋白。此外, 还对抗性相关蛋白的表达特点及基因定量结果进行了分析。该研究结果可为进一步解析抗、感病苹果品种应答轮纹病菌胁迫的抗性机制提供参考。     

Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C.

While searching for a phospholipase C (PLC) specific for phosphatidylcholine in mammalian tissues, we came across such an activity originating from a contamination of Pseudomonas fluorescens. This psychrophilic bacterium was found to contaminate placental extracts upon processing in the cold. The secreted phosphatidylcholine-hydrolyzing PLC was purified by a combination of chromatographic procedures. As substrates, the enzyme preferred dipalmitoyl-phosphatidylcholine and 1-palmitoyl-2-arachidonoyl-phosphatidylcholine over phosphatidylinositol. The active enzyme is a monomer of approximately 40 kDa. As for other bacterial PLCs, the enzyme requires Ca2+ and Zn2+ for activity; dithiothreitol affected the activity due to its chelation of Zn2+, but this inhibition could be compensated for by addition of ZnCl2. The compound D609, described to selectively inhibit phosphatidylcholine-specific PLCs, caused half-inhibition of the P. fluorescens enzyme at approximately 420 microM, while 50-fold lower concentrations similarly affected PLCs from Bacillus cereus and Clostridium perfringens. Partial peptide sequences obtained from the pure P. fluorescens enzyme after tryptic cleavage were used to clone a DNA fragment of 3.5 kb from a P. fluorescens gene library prepared from our laboratory isolate. It contains an ORF of 1155 nucleotides encoding the PLC. There is no significant sequence homology to other PLCs, suggesting that the P. fluorescens enzyme represents a distinct subclass of bacterial PLCs. The protein lacks cysteine residues and consequently contains no disulfide bonds. Interestingly, P. fluorescens reference strain DSMZ 50090 is devoid of the PLC activity described here as well as of the relevant coding sequence.     

Methylobacterium sp. resides in unculturable state in potato tissues in vitro and becomes culturable after induction by Pseudomonas fluorescens IMGB163

Aims:  To induce growth of endophytic bacteria residing in an unculturable state in tissues of in vitro -grown potato plantlets. To isolate and identify the induced bacteria and to localize the strains in tissues of in vitro -grown potato plantlets.
Methods and Results:  The inoculation of in vitro -grown potato plants with Pseudomonas fluorescens IMBG163 led to induction of another bacterium, a pink-pigmented facultative methylotroph that was identified as Methylobacterium sp . using phylogenetic 16S rDNA approach . Two molecular methods were used for localizing methylobacteria in potato plantlets: PCR and in situ hybridization (ISH/FISH). A PCR product specific for the Methylobacterium genus was found in DNA isolated from the surface-sterilized plantlet leaves. Presence of Methylobacterium rRNA was detected by ISH/FISH in leaves and stems of inoculated as well as axenic potato plantlets although the bacterium cannot be isolated from the axenic plants.
Conclusion:  Methylobacterium sp. resides in unculturable state within tissues of in vitro -grown potato plants and becomes culturable after inoculation with P. fluorescens IMBG163.
Significance and Impact of the Study:  In order to develop endophytic biofertilizers and biocontrol agents, a detailed knowledge of the life-style of endophytes is essential. To our knowledge, this is the first report on increase of the culturability of endophytes in response to inoculation by nonpathogenic bacteria.     

Transposon Tn5 mutagenesis of Pseudomonas fluorescens to isolate mutants deficient in antibacterial activity

Abstract Pseudomonas fluorescens was subjected to insertion mutagenesis studies using the transposon Tn5-GM to generate mutants deficient in antibacterial activity minus mutants. The transposon located on the temperature-sensitive plasmid pCHR84 was conjugally transferred into the non-pathogenic pseudomonad using the triparental mating procedure. Random integration of Tn 5 -GM into the chromosome of P. fluorescens was achieved by heat ttreatment of the transformed cells at 42°C. Approximately 2% of transconjugants revealed an auxotrophic phenotype indicating efficient integration of the employed transposon into the chromosome of P. fluorescens . One transposon insertion mutant was obtained showing an antibacterial activity minus phenotype. This mutant (MM-7) was found to be defective in the production of an unidentified antibacterial compound against B. subtilis . These results introduce Tn 5 transposon mutagenesis as a new useful tool for the molecular analysis of P. fluorescens .     

Pseudomonas fluorescens as a potential pathogen: adherence to nerve cells

In order to determine the infectious potential of the psychrotrophic bacterium Pseudomonas fluorescens, a species closely related to the opportunistic pathogen P. aeruginosa, we investigated the binding activity of this bacterium on primary cultures of rat neonate cortical neurons and glial cells, adrenal paraneurons and NG108-15 neuroblastoma cells. Incubated at concentrations of 10(6) and 10(8) CFU/mL, P. fluorescens MF37 exhibited a high binding activity on neurons in the same range as that of P. aeruginosa PAO1. A significant, but lower, adherence of P. fluorescens was also detected on glial cells and adrenal paraneurons. In contrast, when P. fluorescens MF37 or P. aeruginosa PAO1 were incubated with neuroblastoma cells, no binding was observed. In neurons, the association of P. fluorescens with the plasma membrane occurred both on neurites and cell body. Leakage of the cytoplasmic content was frequently noted. Studies performed using the fluorescent probe Hoechst 33258 revealed that in 10% of neurons, P. fluorescens induced the appearance of densely stained clusters of DNA that was typical of an early step of apoptosis. In glial cells exposed to P. fluorescens, marked changes in the morphology of the nucleus, including fragmentation into lobular structures and aggregation of DNA, were also reminiscent of the existence of a possible apoptotic mechanism. Taken together, these results reveal that P. fluorescens can bind to nerve cells and affect their physiology and, in agreement with recent clinical observations, suggest that P. fluorescens could behave as a pathogen.     

硒在苹果叶片中的赋存形式

通过筛选‘红富士’苹果适宜的叶面施硒浓度,研究了营养元素硒在苹果叶片中的赋存形式、分布及含硒大分子的提取分离技术。结果表明,（1）‘红富士’苹果最佳叶面施硒浓度为100mg·L-1,此处理的叶片总硒含量为2．7283mg·L-1（FW）,其中有机硒含量为2．3384mg·L-1（FW）,有机化程度达85．71％。（2）无论在对照组还是处理组,叶片中的有机硒均主要以蛋白质结合态存在,平均占有机硒的77．09％。（3）蛋白质、多糖和核酸3种有机物在100mg·L-1最优处理组的叶片中结合硒的量分别占有机硒含量的77．42％、18．46％和0．54％。（4）在100mg·L-1处理组的蛋白质组分中,又以盐溶性蛋白质结合硒的量最多,占蛋白质结合态硒含量的46．27％。     

Methode zum Nachweis der Dauersporen des Kohlhernieerregers Plasmodiophora brassicae in gärtnerischen Kultursubstraten

Populations of pathogenic Pseudomonas syringae pv. syringae were monitored on apparently healthy leaves, blossoms, and fruit from two apple orchards with known histories of blister bark and a pear orchard with a known history of blossom blast. Populations on blossoms and fruits were higher on pears than on apples. Yellow-pigmented, non-pathogenic bacteria might have suppressed or masked the presence of P. syringae pv. syringae on apple trees. Populations of P. syringae pv. syringae on apple and pear leaves fluctuated sharply but higher levels generally occurred during the 1984/85 growing season than during the drier 1983/84 season. This investigation indicates that the resident phase of P. syringae pv. syringae is probably a major source of inoculum for apple blister bark and pear blossom blast in South Africa.     

The effects of glutaraldehyde on the control of single and dual biofilms of Bacillus cereus and Pseudomonas fluorescens

Glutaraldehyde (GLUT) was evaluated for control of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens on stainless steel surfaces using a chemostat system. The biofilms were characterized in terms of mass, cell density, total and matrix proteins and polysaccharides. The control action of GLUT was assessed in terms of inactivation and removal of biofilm. Post-biocide action was characterized 3, 7, 12, 24, 48 and 72 h after treatment. Tests with planktonic cells were also performed for comparison. The results demonstrated that in dual species biofilms the metabolic activity, cell density and the content of matrix proteins were higher than those of either single species. Planktonic B. cereus was more susceptible to GLUT than P. fluorescens. The biocide susceptibility of dual species planktonic cultures was an average of each single species. Planktonic cells were more susceptible to GLUT than their biofilm counterparts. Biofilm inactivation was similar for both of the single biofilms while dual biofilms were more resistant than single species biofilms. GLUT at 200 mg l(-1) caused low biofilm removal (<10%). Analysis of the post-biocide treatment data revealed the ability of biofilms to recover their activity over time. However, 12 h after biocide application, sloughing events were detected for both single and dual species biofilms, but were more marked for those formed by P. fluorescens (removal >40% of the total biofilm). The overall results suggest that GLUT exerts significant antimicrobial activity against planktonic bacteria and a partial and reversible activity against B. cereus and P. fluorescens single and dual species biofilms. The biocide had low antifouling effects when analysed immediately after treatment. However, GLUT had significant long-term effects on biofilm removal, inducing significant sloughing events (recovery in terms of mass 72 h after treatment for single biofilms and 42 h later for dual biofilms). In general, dual species biofilms demonstrated higher resistance and resilience to GLUT exposure than either of the single species biofilms. P. fluorescens biofilms were more susceptible to the biocide than B. cereus biofilms.     

Expression of the Pseudomonas syringae avirulence protein AvrB in plant cells alleviates its dependence on the hypersensitive response and pathogenicity (Hrp) secretion system in eliciting genotype-specific hypersensitive cell death.

The nonpathogenic bacteria Pseudomonas fluorescens and Escherichia coli can elicit a genotype-specific hypersensitive response (HR) in plants if they express both the HR and pathogenesis (Hrp) protein secretion system and the HrpZ harpin from P. syringae pv syringae 61 and a P. syringae avirulence (avr) gene whose presence is recognized by a corresponding disease resistance gene in the plant. We have found that the recognition event appears to require transfer of the Avr protein into the plant cell. Elicitation of a genotype-specific HR was observed with avrB+ P. fluorescens in soybean and Arabidopsis plants carrying resistance genes RPG1 and RPM1, respectively, and with avrPto+ E. coll in tomato plants carrying resistance gene PTO, but only if the Hrp secretion system, HrpZ, and the appropriate Avr proteins were produced in the same bacterial cell. The failure of avrB hyperexpression and exogenous AvrB or HrpZ to alleviate these requirements in soybean and Arabidopsis suggests that the site of AvrB action is not in the bacterial cell or plant apoplast. An Arabidopsis rps3 (rpm1) glabrous1 mutant was transformed with constructs expressing avrB and was crossed with an Arabidopsis ecotype Columbia (RPM1 GLABROUS1) plant. F1 seedlings (identified by their kanamycin-resistant, pubescent phenotype) exhibited extensive necrosis on cotyledon leaves 10 days postgermination. Ecotype Columbia and rps3-1 leaves biolistically cobombarded with plasmids expressing the beta-glucuronidase (GUS) gene and avrB failed to produce GUS activity (indicative of cell death) only when RPM1 and avrB were present in the leaf. Thus, both stable and transient expression of avrB in Arabidopsis resulted in RPM1-dependent necrosis, and the only demonstrable site of action for AvrB was inside plant cells.