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Background Agrobacterium rhizogenes-mediated (ARM) transformation is a highly efficient technique for generating composite plants composed of transgenic roots and wild-type shoot, providing a powerful tool for studying root biology. The ARM transformation has been established in many plant species, including soybean. However, traditional transformation of soybean, transformation efficiency is low. Additionally, the hairy roots were induced in a medium, and then the generated composite plants were transplanted into another medium for growth. This two-step operation is not only time-consuming, but aggravates contamination risk in the study of plant-microbe interactions. Results Here, we report a one-step ARM transformation method with higher transformation efficiency for generating composite soybean plants. Both the induction of hairy roots and continuous growth of the composite plants were conducted in a single growth medium. The primary root of a 7-day-old seedling was decapitated with a slanted cut, the residual hypocotyl (maintained 0.7-1 cm apical portion) was inoculated with A. rhizogenes harboring the gene construct of interest. Subsequently, the infected seedling was planted into a pot with wet sterile vermiculite. Almost 100% of the infected seedlings could produce transgenic positive roots 16 days post-inoculation in 7 tested genotypes. Importantly, the transgenic hairy roots in each composite plant are about three times more than those of the traditional ARM transformation, indicating that the one-step method is simpler in operation and higher efficiency in transformation. The reliability of the one-step method was verified by CRISPR/Cas9 system to knockout the soybean Rfg1, which restricts nodulation in Williams 82 (Nod-) by Sinorhizobium fredii USDA193. Furthermore, we applied this method to analyze the function of Arabidopsis YAO promoter in soybean. The activity of YAO promoter was detected in whole roots and stronger in the root tips. We also extended the protocol to tomato. Conclusions We established a one-step ARM transformation method, which is more convenient in operation and higher efficiency (almost 100%) in transformation for generating composite soybean plants. This method has been validated in promoter functional analysis and rhizobia-legume interactions. We anticipate a broad application of this method to analyze root-related events in tomato and other plant species besides soybean.

Additional file 4: Figure S4. One-step generation of composite tomato plant with transgenic roots by ARM transformation. Histochemical analyses of tomato hairy roots transformed with K599 harboring pCAMBIA1305 where contains GUS reporter gene driven by CaMV35S promoter. Tomato (Solanum lycopersicum) seeds Maofen 802 were used.

Additional file 2: Figure S2. CRISPR/Cas9-mediated knockout of Rfg1 in the Williams 82 (Nod-). Mature fixation nitrogen root nodules formed on a transgenic root (pointed out by arrows)

Additional file 3: Figure S3. A sequencing identification from CRISPR/Cas9-mediated knockout of Rfg1 in the Williams 82 (Nod–) background. One targeted knockout site (indicated in the black box) in Williams 82 wild type background and its flanking sequence. The protospacer adjacent motif (PAM) is ‘TGG’ (a). Sequencing analysis of the DNA from transgenic nodules revealed that two mutant alleles were caused, one with a 152-bp deletion (b) and another with a 70- bp deletion (c).

Additional file 5: Figure S5. Maps and sequences of the binary vector. pGSE401-KtRfg1vector. The targeted site was highlighted in the red background (a); pCAMBIA1305 vector (b); pYGUS1305 (c).

Additional file 9: Table S1. A comparison of hairy roots produced by one-step and two-step method.

Additional file 1: Figure S1. The T-DNA insertion sites in the soybean genome determined by sequencing of TAIL-PCR products. Three examples of sequencing analysis (partial sequences are indicated) of the TAIL-PCR products from three different independent transgenic hairy roots (in A, B, and C, respectively). Chr. 13 (in panel A), Chr. 20 (in panel B), Chr. 2 (in panel C) represented chromosome number. The gray filled triangles indicated the T-DNA insertions and the numbers showed the positions of the insertion site. Green highlighted letters indicated the T-DNA sequences of the right border side from pCAMBIA1305.1. Yellow highlighted letters indicated genomic flanking sequences of T-DNA insertion.

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most devastating bacterial disease of rice (Oryza sativa L.), a staple food crop that feeds half of the world’s population. In management of this disease, the most economical and effective approach is cultivating resistant varieties. Due to rapid change of pathogenicity in the pathogen, it is necessary to identify and characterize more host resistance genes for breeding new resistant varieties. We have previously identified the BB resistance (R) gene Xa23 that confers the broadest resistance to Xoo strains isolated from different rice-growing regions and preliminarily mapped the gene within a 1.7 cm region on the long arm of rice chromosome 11. Here, we report fine genetic mapping and in silico analysis of putative candidate genes of Xa23. Based on F2 mapping populations derived from crosses between Xa23-containing rice line CBB23 and susceptible varieties JG30 or IR24, six new STS markers Lj36, Lj46, Lj138, Lj74, A83B4, and Lj13 were developed. Linkage analysis revealed that the new markers were co-segregated with or closely linked to the Xa23 locus. Consequently, the Xa23 gene was mapped within a 0.4 cm region between markers Lj138 and A83B4, in which the co-segregating marker Lj74 was identified. The corresponding physical distance between Lj138 and A83B4 on Nipponbare genome is 49.8 kb. Six Xa23 candidate genes have been annotated, including four candidate genes encoding hypothetical proteins and the other two encoding a putative ADP-ribosylation factor protein and a putative PPR protein. These results will facilitate marker-assisted selection of Xa23 in rice breeding and molecular cloning of this valuable R gene.

We have previously isolated a Brassica juncea cDNA encoding a novel chitinase BjCHI1 with two chitin-binding domains (Zhao and Chye in Plant Mol Biol 40:1009-1018, 1999). The expression of BjCHI1 was highly inducible by methyl jasmonate (MeJA) treatment, wounding, caterpillar feeding, and pathogenic fungal infection. These observations suggest that the promoter of BjCHI1 gene might contain specific cis-acting elements for stress responses. Here, we report the cloning and characterization of the BjCHI1 promoter. A 1,098 bp BjCHI1 genomic DNA fragment upstream of the ATG start codon was isolated by PCR walking and various constructs were made by fusing the BjCHI1 promoter or its derivatives to beta-glucuronidase reporter gene. The transgenic Arabidopsis plants showed that the BjCHI1 promoter responded to wounding and MeJA treatment, and to treatments with either NaCl or polyethyleneglycol (PEG 6000), indicating that the BjCHI1 promoter responses to both biotic and abiotic stresses. A transient gene expression system of Nicotiana benthamiana leaves was adopted for promoter deletion analysis, and the results showed that a 76 bp region from -695 to -620 in the BjCHI1 promoter was necessary for MeJA-responsive expression. Furthermore, removal of a conserved T/G-box (AACGTG) at -353 to -348 of the promoter greatly reduced the induction by MeJA. This is the first T/G-box element identified in a chitinase gene promoter. Gain-of-function analysis demonstrated that the cis-acting element present in the 76 bp region requires coupling with the T/G-box to confer full magnitude of BjCHI1 induction by MeJA.

Xanthomonas oryzae pv. oryzae causes bacterial blight of rice. Xa23, a bacterial blight resistance gene identified originally in wild rice, Oryza rufipogon, is dominant and resistant to all X. oryzae pv. oryzae field isolates tested. The corresponding avirulence gene avrXa23 is unknown. Here we report the generation of a random insertion mutant library of X. oryzae pv. oryzae strain PXO99 using a Tn5-derived transposon tagging system, and identification of mutant strains that are virulent on CBB23, a near-isogenic rice line containing Xa23. A total of 24,192 Tn5 inserted clones was screened on CBB23 by leaf-cutting inoculation and at least eight of them caused lesions on CBB23 comparable to those on JG30, the susceptible recurrent parent of CBB23. Polymerase chain reaction and Southern blot analysis showed that all the eight mutants, designated as P99M1, P99M2, P99M3, P99M4, P99M5, P99M6, P99M7 and P99M8, have a single Tn5-insertion in their genomes. The flanking DNA sequences of the Tn5-insertion sites were isolated by PCR-walking and sequenced. Bioinformatic analysis of the flanking sequences, by aligning them with the whole genome sequences of X. oryzae pv. oryzae strains PXO99, KACC10331 and MAFF311018 through NCBI, revealed that the Tn5-insertions disrupted genes that encode TAL effector AvrBs3/PthA, ISXo1 transposase, Type II secretion system protein-like protein or outer membrane protein, glycogen synthase, cytochrome C5 and conserved hypothetical protein. Further identification of these mutants will facilitate the molecular cloning of avirulence gene avrXa23.

GA 20-oxidase (GA20ox) is a regulatory enzyme for the syntheses of biologically active GAs in plants. The loss-of-function mutations in OsGA20ox2 of rice (Oryza sativa L.) generate the well-known Green Revolution gene sd-1, which cause the semi-dwarfism phenotype. In our present investigation, semi-dwarf plants were generated from a taller rice variety QX1 by RNAi suppression on the expression of OsGA20ox2. The 531bp-fragment of OsGA20ox2 was amplified by PCR from genomic DNA of QX1 and used to construct the hairpin RNAi vector pCQK2. The wild type QX1 was transformed with pCQK2 by Agrobacterium-mediated transformation and some independent transgenic RNAi lines exhibited semi-dwarfism. RT-PCR and Northern blot analyses showed that the expression of OsGA20ox2 was specifically suppressed in the RNAi semi-dwarf lines. Endogenous GA assays revealed that the contents of the GA20ox2-catalyzed products GA19, GA20 and the down-stream biologically active GA1 were drastically reduced in the RNAi semi-dwarf lines. We further showed that the RNAi semi-dwarf lines could be restored to normal plant height by applying exogenous GA3. The results indicated that the semi-dwarfism of the RNAi semi-dwarf lines was associated with the decreased expression of OsGA20ox2 gene and the reduced content of endogenous biologically active GA1. Analyses of panicle length, seeds per panicle and 1000-grain weight suggested that the RNAi semi-dwarf lines showed stable grain yield compared with the wild type plants. It is demonstrated that the RNAi approach could be useful for plant breeding purposes in the future.

The majority of plant disease resistance (R) genes encode proteins that share common structural features. However, the transcription activator-like effector (TALE)-associated executor type R genes show no considerable sequence homology to any known R genes. We adopted a map-based cloning approach and TALE-based technology to isolate and characterize Xa23, a new executor R gene derived from wild rice (Oryza rufipogon) that confers an extremely broad spectrum of resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Xa23 encodes a 113 amino acid protein that shares 50% identity with the known executor R protein XA10. The predicted transmembrane helices in XA23 also overlap with those of XA10. Unlike Xa10, however, Xa23 transcription is specifically activated by AvrXa23, a TALE present in all examined Xoo field isolates. Moreover, the susceptible xa23 allele has an identical open reading frame of Xa23 but differs in promoter region by lacking the TALE binding element (EBE) for AvrXa23. XA23 can trigger a strong hypersensitive response in rice, tobacco, and tomato. Our results provide the first evidence that plant genomes have an executor R gene family of which members execute their function and spectrum of disease resistance by recognizing the cognate TALEs in the pathogen.