Characterization of T-DNA insertion patterns in the genome

Curr Genet (2007) 51:233–243DOI 10.1007/s00294-007-0122-5RESEARCH ARTICLECharacterization of T-DNA insertion patterns in the genomeof rice blast fungus Magnaporthe oryzaeGuihua Li · Zhuangzhi Zhou · Guifu Liu ·Fucong Zheng · Chaozu HeReceived: 11 December 2006 / Revised: 27 January 2007 / Accepted: 29 January 2007/ Published online: 16 February 2007© Springer-Verlag 2007Abstract Agrobacterium tumefaciens-mediated trans-formation (ATMT) has been proven to be a powerful strategy for gene disruption in plants and fungi. Pat-terns associated with transferred DNA (T-DNA) integration in plants and yeast have been studied comprehensively, whereas no detailed analysis of T-DNA integration has been reported yet in W lamen-tous fungi. Here, we reported the T-DNA insertion patterns in the genome of W lamentous fungus Magna-porthe oryzae. Using ATMT, a T-DNA tagged popula-tion consisting of 6,179 transformants of M. oryzae was constructed. With thermal asymmetric interlaced-PCR (TAIL-PCR), 623 right border (RB) X anking sequences and 124 left border (LB) X anking sequences were generated. Analysis of these X anking sequences indicated a signi W cant integration bias toward non-cod-ing sequences, suggesting distribution of T-DNAs was not random. Comparing to T-DNA RB, LB was nicked inaccurately and truncated frequently during integration. Chromosomal rearrangements, such as deletion, inversion, and translocation, were associated with T-DNA integration in some transformants. Our data suggest that, comparing with plant cells, T-DNA integrates into this W lamentous fungus with more pre-cise and simpler patterns. Some phenotypic mutants were observed in our T-DNA tagged population, and these transformants will be very useful for functional genomics research of M. oryzae.Keywords Agrobacterium tumefaciens-mediated transformation · Magnaporthe oryzae · T-DNA · Chromosomal rearrangementIntroductionMagnaporthe oryzae (formerly Magnaporthe grisea), the causative agent of rice blast, is the most destructive pathogen of rice worldwide (Zeigler et al. 1994) and the principal model organism for elucidating the molecular basis of fungus-plant interactions (Valent 1990). M. oryzae is a haploid ascomycete fungus and contains seven chromosomes in each cell. The draft genome sequence of M. oryzae strain 70-15 has been released (Dean et al. 2005). Functional and com-parative genomic analysis of this important phytopath-ogenic fungus, although still in the early stage, has improved our understanding of the molecular mecha-nisms involved in host-pathogen interactions (Xu et al. 2006). Large-scale expressed sequence tags (EST), whole-genome microarrays and mutant collections have been developed (Soanes et al. 2002; Xu et al. 2006). Among available approaches for W nding novel genes that encode determinants of pathogenicity,Communicated by K. Borkovich.Guihua Li and Zhuangzhi Zhou contributed equally to the work.Electronic supplementary material The online version of this article (doi:10.1007/s00294-007-0122-5) contains supplementary material, which is available to authorized users.G. Li · Z. Zhou · G. Liu · F. Zheng · C. He (&)State Key Laboratory of Plant Genomics,Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People’s Republic of Chinae-mail: hecz@G. Li · Z. ZhouGraduate School of the Chinese Academy of Sciences,Beijing 100039, People’s Republic of Chinaidenti W cation of pathogenicity-de W cient mutants clearly has the greatest potential. Agrobacterium tumefaciens-mediated transformation (ATMT) has been proved to be an e V ective way for this aim in plants. Transferred DNA (T-DNA) transformation of M. oryzae was demonstrated successfully recently (Rho et al. 2001).Agrobacterium tumefaciens is able to transfer its T-DNA into diverse groups of organisms, including plants, other prokaryotes, yeast, W lamentous fungi, even human cells, which represents a unique case of naturally occurring trans-kingdom DNA transfer (Lac-roix et al. 2006). Whereas extensive work has revealed the translocation process, little is known about the bio-logical mechanisms governing the targeting of the T-DNA to its site of integration and following insertion within the host chromatin. Comprehensive character-izations associated with T-DNA insertion into plants have been reported recently, especially in Arabidopsis, tobacco, and rice (Tz W ra et al. 2004).Transferred DNA integrates into plant cells mostly with a non-homologous recombination mode (Gheysen et al. 1991; Mayerhofer et al. 1991), and a signi W cant bias of integration events in 5Ј-up-stream (5Ј untranslated region and promoter) regions of genes has been dis-closed (Alonso et al. 2003; Chen et al. 2003; Forsbach et al. 2003; Sha et al. 2004). At regions surrounding centromeres of Arabidopsis, T-DNA integration events are observed rarely (Alonso et al. 2003).In Agrobacterium, the 25bp T-DNA border repeat sequence is predominantly nicked at the T-DNA lower strand, between the third and fourth nucleotide (Sta-chel et al. 1986; Wang et al. 1987). T-DNA integrates into plant cells with a more canonical right border (RB) than left border (LB) (Brunaud et al. 2002; Fors-bach et al. 2003; Kim et al. 2003), probably due to the protection of VirD2 protein which has been attached to RB for most of the time during the process of T-DNA transfer (Tinland et al. 1995; Brunaud et al. 2002).Transferred DNA insertions in Arabidopsis (Ghey-sen et al. 1991; Mayerhofer et al. 1991; Forsbach et al. 2003) and tobacco (Ohba et al. 1995) are normally associated with small (<100bp) target site deletions. Chromosomal rearrangements such as inversion and translocation, associated with T-DNA insertions, are also observed in transgenic Arabidopsis (Castle et al. 1993; Nacry et al. 1998; Forsbach et al. 2003) with low frequency.At the junction between T-DNA and the plant genomic DNA, W ller sequence (Forsbach et al. 2003; Windels et al. 2003) and microhomology are frequently observed, whereas the frequency of microhomology is found higher at the junctions involving LB than those of RB (Forsbach et al. 2003; Kim et al. 2003).Long T-DNA (T-DNA and non-T-DNA) transfer is a very common phenomenon occurred during the process of ATMT of plants (Virts and Gelvin 1985; Wenck et al. 1997; Yin and Wang 2000; Sha et al. 2004). Insertions with a size of greater-than-unit-length of the binary plasmid have been detected in transgenic Arabidopsis (Wenck et al. 1997) and rice (Yin and Wang 2000). Based on analysis of co-trans-formation with two di V erent T-DNAs containing dis-tinct sequences adjacent to T-DNA borders, it is concluded that T-DNAs frequently integrate into same positions of plant genomes with inverted- or direct-repeat modes (De Neve et al. 1997; De Buck et al. 1999).Recent studies indicate that T-DNA integration into double-stranded break (DSB) with a double-stranded form might be a default mode of T-DNA insertion in host genomes (Köhler et al. 1989; Chilton and Que 2003; Tz W ra et al. 2003; Tz W ra et al. 2004).Agrobacterium tumefaciens-mediated transforma-tion has been established in many W lamentous fungi including M. oryzae (Michielse et al. 2005). However, patterns of T-DNA insertion in these fungal genomes are not clear. In this paper, the T-DNA integration patterns into the genome of M. oryzae are explored. We constructed a T-DNA tagged population of M.oryzae containing 6,179 transformants, from which 623 RB X anking sequences and 124 LB X anking sequences were rescued and analyzed. Our results showed that distribution of T-DNAs among seven chromosomes of M. oryzae was not random, and T-DNA integration could induce chromosomal rear-rangements such as deletion, inversion, and transloca-tion. Our data suggest that T-DNA integrates into the genome of M. oryzae with a more precise and simpler mode than that into plant cells, which provides advan-tages for study of functional genomics of the W lamen-tous fungus.Materials and methodsFungal strains and culture conditionsTwo strains of M. oryzae were used in this study, a Chinese W eld strain Y34 was used in ATMT and a laboratory strain 70-15 was used as a reference strain. The fungus was cultured on oatmeal agar (OMA) at 28°C. For conidiation, the 5–7days plate culture was scratched, followed by additional 3–4days cultivation under continuous X uorescent light.Agrobacterium-mediated transformationof Magnaporthe oryzaeAgrobacterium tume W ciens strain Agl-1, carrying the binary vector pBHt2 with hpt (hygromycin phospho-transferase) gene under the Aspergillus nidulans trpC promoter (Mullins et al. 2001), was used to transform strain Y34 with the protocol as previously described (Rho et al. 2001). Hygromycin B (200 g/ml) was used for selection of transformants.Mitotic stability of T-DNA inserted in the Magnaporthe oryzae genomeFive randomly selected transformants were cultured on OMA plates without hygromycin B for 4–5days. Mycelia at the edge of the culture was picked up with toothpick and inoculated on fresh OMA plates for growth of another 4–5days. After repeating this proce-dure for 16 times, germinating monoconidium from each transformant was transferred to OMA plate con-taining hygromycin B (200 g/ml) and their growth was observed. DNAs of each strain were extracted for Southern blot analysis with the probe ampli W ed with primers HPTa and HPTb (Supplementary Table S1), which were speci W c to hpt gene and with an 858bp ampli W cation product.DNA isolation and analysisFollowing growth on OMA plates for 4–7days, mycelia of each strain were collected and cultured in V-8 juice media with hygromycin B (75 g/ml) and streptomycin (100 g/ml) for additional 2–4days. Then, mycelia were harvested by vacuum-in W ltration and washed by sterile water. Mycelia were ground to W ne powders in liquid nitrogen. Genomic DNA was isolated as previously described (Talbot et al. 1993).For Southern blot analysis, about 1 g of genomic DNA was digested with restriction endonucleases, sep-arated in 1% agarose gels, blotted to Hybond-N+ mem-branes and hybridized with [32P]dCTP-labeled probe. Hybridization and washes were performed using stan-dard procedures (Sambrook et al. 1989).Isolation of T-DNA X anking sequencesThermal asymmetric interlaced-PCR (TAIL-PCR) (Liu and Whittier 1995; Mullins et al. 2001) was employed to obtain genomic DNA sequences X anking inserted T-DNAs from transformants selected ran-domly. Primers designed for TAIL-PCR were list in Supplementary Table S1. The tertiary TAIL-PCR products were cloned into pGEM-T Easy vector (Pro-mega, Madison, WI, USA) and sequenced. Sequence processingSequences of TAIL-PCR products were searched for nucleotide similarities by the BLASTN algorithm against databases as follows: M. oryzae genome database (/annotation/genome/ magnaporthe_grisea) and NCBI (http://www.ncbi.nlm. /BLAST). Positions of the T-DNA insertion sites were mapped in silico onto M. oryzae chromo-somes based on the physical and genetic maps available at the M. oryzae genome database.Distribution of T-DNA insertion sites among seven chromosomes was analyzed according to length of each linkage group released from the latest Assembly Struc-ture of the M. oryzae genome database. Distribution of T-DNA insertion sites within particular genetic ele-ments was analyzed according to data derived from Genome Statistics of the M. oryzae genome database. Searches for 1kb up-stream or 200bp down-stream of annotated genes of M. oryzae were preformed for dis-tribution of T-DNA insertion sites in the 5Ј- or 3Ј-regu-latory regions.Nicking sites of T-DNA integrations were deter-mined by comparison of each end sequence of the inte-grated T-DNA, which was revealed by X anking sequence analysis, with the end sequence of canonical one.Plasmid rescueAbout 1 g of genomic DNA of each tested transfor-mant was digested with restriction endonuclease Pst I, which has only one digestion site in plasmid pBHt2 at its multiple cloning site. The digested DNA was etha-nol precipitated, dissolved in sterile water and ligated using T4 DNA ligase at 16°C overnight. The product of ligation was precipitated, redissolved in 20 l sterile water and electroporated into Escherichia coli DH10B cells. Transformants were cultured on LB plates with kanamycin (50 g/ml) for plasmid selection.Analysis of chromosomal rearrangementEach putative transformant, involving rearrangement probably revealed by TAIL-PCR, was further analyzed by PCR using four sets of primer pairs designed according to the surrounding sequences of junction sites (primers LF1-3 and LR1-3 for LB junction sites, RF1-3 and RR1-3 for RB junction sites, Supplemen-tary Table S1). PCR products containing host–hostjunction sites were sequenced to verify the rearrange-ments. In order to further con W rm chromosomal inver-sion occurred in a transformant, Southern blot analysis with six probes derived from rearranged chromosomal regions was carried out.ResultsDetermination of T-DNA copy number and mitotic stability of transformants of Magnaporthe oryzaeAn ATMT system of M. oryzae with transformation e Y ciency about 300 transformants per 1£106 conidia, was established, from which 6,179 independent trans-formants were obtained. To determine T-DNA inte-gration and copy number, 135 transformants were selected randomly. PCR analysis with primers HPTa and HPTb indicated that all tested transformants con-tained Hpt gene (data not shown). Genomic DNAs of these transformants were analyzed by Southern blot probed with a fragment of Hpt gene (Fig.1). The result indicated that 72.6% (98/135) of the transformants contained one copy of T-DNA, 19.2% (26/135) con-tained two copies, 8.2% (11/135) contained three or more copies. The average copy number of T-DNA integrated into these transformants was 1.36. Five ran-domly selected transformants were assayed for their mitotic stability of inserted T-DNA. These trasnfor-mants remained their hygromycin B resistance and T-DNA copy number in subsequent sub-culture for 16 times in the absence of hygromycin B, indicating that T-DNA was stable in the genome of M. oryzae. Analysis of T-DNA X anking sequencesThermal asymmetric interlaced-PCR was carried out to recover X anking sequences of inserted T-DNAs. Excluding the non-speci W c or similar, we obtained 623 RB X anking sequences from 900 transformants and 124 LB X anking sequences from 240 transformants, respec-tively. From them, genomic sequences adjacent to both borders of a same T-DNA were recovered from 75 transformants. Of these 623 RB X anking sequences, 445 corresponded to single copy genomic regions; 16 matched to multiple genomic regions, suggesting that the corresponding T-DNAs were integrated into repet-itive sequence regions; 150 X anking sequences con-tained sequences derived from binary vector backbone or another T-DNA (of which M. oryzae sequences could be found in three X anking sequences); and 14 X anking sequences were not matched to any sequences of M. oryzae or binary vector pBHt2, which might locate in un-sequenced regions.Distribution of T-DNA insertion sites among seven chromosomesThe 445 solely matched X anking sequences were analyzed for distribution of T-DNA insertion sites. Of which, 432 X anking sequences were mapped on seven chromosomes, respectively, while 13 X anking sequences located on unlinked surpercontigs (Fig. 2, Supplementary Table S2). According to length of each chromosome available at the M. oryzae genome data-base, expected frequencies of T-DNA integrations into seven chromosomes from I to VII based on random insertion are 21.0, 16.0, 15.8, 10.6, 13.9, 11.7, and 11.0%, respectively. Expected T-DNA numbers dis-tributed among seven chromosomes were calculated for the mapped 432 X anking sequences (Table1). The results revealed that the distribution of T-DNA inser-tion sites among seven chromosomes was not com-pletely random. Although no signi W cant bias (at P=0.05 level) of T-DNA integration events into chro-mosomes II, IV, V, VI, and VII were observed, T-DNA insertions toward chromosome I appeared more frequent than expected. However, for chromosome III, the frequency of T-DNA integration was signi W cantly lower than expected. Fewer insertion events of T-DNA were found at some inner regions where centromeres might locate (Fig.2).Fig.1Southern blot analysis of transformants of Magnaportheoryzae. Genomic DNAs of di V erent transformants and wild type strain Y34 were digested with Pst I and hybridized with a fragment of hpt gene. Lane 1, 1kb DNA marker (New England Biolabs); lanes 2–31, di V erent transformants; lane 32, wild type strain Y34Distribution of T-DNA insertion sites within particular genetic elementsGenetic elements were deduced from gene predictions of the latest release of the M. oryzae genome annota-tion (Version 5.0). In the genome, a gene was found, on average, every 3,241.5bp including 1,549bp of genic sequences (1,319bp in exons and 230bp in introns).Assuming completely random T-DNA integration we would expect 47.8% of the insertions in CodingFig.2Scheme of distribution of T-DNAs in the genome of Magnaporthe oryzae .Chromosomal positions of 432 T-DNA RB X anking sequences were determined according to the physical and genetic maps available at the Magnaporthe oryzae genome database. The loci of corresponding T-DNApositions were shown on left sides of each chromosome. Genetic markers ofMagnaporthe oryzae were shown on rightTable 1Statistical analysis of the distribution of T-DNAs among seven chromosomesExact integration sites of these T-DNAs can be found in Supplementary Table S22 values were calculated for 432 T-DNAs, which map positions on chromosomes were determined unambiguously *, ** Indicate signi W cance for 2 test at P =0.05 and 0.01 levels, respectivelyChromosomes TotalIII III IV V VI VII Expected T-DNA number 90.7569.0768.0945.6760.0850.5847.76432Observed T-DNA number109*6948**40675346432sequences (40.7% in exons and 7.1% in introns), 30.8%in the region 1kb up-stream of a start codon, and 6.2%in the region 200bp down-stream of a stop codon. The remainder (15.2%) should insert in intergenic regions. 2 tests (P =0.01) indicated that T-DNA insertions in genic sequences were found less frequently than expected, mostly due to the low frequency of insertion in exon. However, a signi W cant insertion bias was observed in favor of non-genic sequences, especially up-stream 1kb regions and intergenic regions. No sig-ni W cant integration biases were observed in introns or down-stream 200bp regions of annotated genes (Table 2).Integration accuracy of T-DNA bordersFive hundred and W fty-eight RB X anking sequences (of 623, the remainder 65 X anking sequences belong to long T-DNA integration) and 124 LB X anking sequences were analyzed for integration accuracy of T-DNA borders (Fig.3). The results showed that the accuracy of integration was very high at RB. In 92.1%(514 of 558 X anking sequences) of the cases, T-DNAs integrated with canonical RBs (i.e., nicking positions appear between the third and fourth nucleotide of the 25bp border repeat sequence), while deletions with small fragment (1–80bp) were only found in 7.9% (44of 558 X anking sequences) of the cases. Compared withRB, however, the accuracy of LB integration was low.Only 32.3% (41 of 124) were canonical insertions. T-DNA integrations with LBs truncated with 1–22bp were observed frequently (59.7%, 73 of 124). Large deletions (30–150bp) occurred in lower frequency (8.1%, 10 of 124).Microhomologies and W ller sequences observed at the junctions between the host genome and T-DNA bordersThe 124 LB and the 462 RB X anking sequences, which contained sequences of M. oryzae , were analyzed for patterns of junctions between T-DNA borders and genomic target sites (Table 3). Microhomologies with 2–7bp in size were found frequently (72.6%, 90 of 124)in junctions between LB and genomic sequences. How-ever, in junctions involving RB, microhomologies were found less frequently (21.0%, 97 of 462) and mostly in smaller size (1bp). Meanwhile, W ller sequences ranging from 1 to 230bp in size were observed from 56 of 462(12.1%) junctions of RB. Sequence analysis showed that, some longer W ller sequences were originated from the T-DNA region, backbone of binary vector or the host genome, respectively. It was di Y cult to determine the originations of the shorter W ller sequences. At the junctions of LB, W ller sequences occurred rarely (1.6%,2 of 124).Sequence analysis of T-DNA insertion sites and chromosomal rearrangements associated with T-DNA integrationFrom RB and LB X anking sequences mentioned above, genomic sequences adjacent to both borders of a same T-DNA were obtained from 75 transformants.These X anking sequences were compared with sequences corresponding to pre-insertion sites (Table 4).The results indicated that, deletions with 1–80bp in size (15bp in average) were observed frequently (60 of 75)in genomic target sites due to T-DNA integration. The largest deletion with 505bp in size was found in oneTable 2Distribution of T-DNA insertion sites within particular genetic elements ** Indicates signi W cance at P =0.01 level for 2 testsPosition of insertion Expected number of T-DNA Observed number of T-DNA Non-genic sequences 232.35364**Up-stream 1,000bp 137.28186**Down-stream 200bp 27.4620Intergenic sequences 67.61159**Genic sequences 212.6580**Exon 181.0654**Intron31.5926Total sequences analyzed445Fig.3Nicking positions of the integrated T-DNAs.Histogram of the percentage of nicking site was classi W ed based on the nicking fre-quency calculated from 124 LB or 558 RB X anking se-quences, respectively. T-DNA border repeats were capital-ized. Arrows indicate the posi-tions of T-DNA nicking in Agrobacteriumtransformant. Short duplications (1–8bp) of the target sequences were also observed in W ve T-DNA insertion events. Exact integration without deletion or duplica-tion was only found in six transformants. These results suggest that small fragment deletion of the target site is the most frequent event during T-DNA integration into the genome of M. oryzae.Three single-copy T-DNA transformants (6001, 4825, and 438), which had RB and LB X anking sequences localized on di V erent chromosomes, or on the same chromosome but far away from each other, were identi W ed, which suggested that chromosomal rearrangement might occur. PCR was applied to fur-ther analysis, with primers (Supplementary Table S1) speci W c to corresponding target sites of each transfor-mant. Ampli W ed products were only obtained from genomic DNA templates of wild type strains using primer pairs LF+LR (LF1+LR1, LF2+LR2, and LF3+LR3 for transformant 6001, 4825, and 438, respectively) or RF+RR (RF1+RR1, RF2+RR2, and RF3+RR3 for transformant 6001, 4825 and 438, respectively) (Fig.4a–c). However, when primer pairs LF+RR (LF1+RR1, LF2+RR2, and LF3+RR3, respectively) or RF+LR (RF1+LR1, RF2+LR2, and RF3+LR3, respectively) which had annealing sites on di V erent fragments involved in rearrangement,were used, PCR products with predicted sizes were ampli W ed from all three transformants, while no prod-uct was obtained from wild type strains (Fig.4a–c). These results demonstrated that chromosomal rear-rangements induced by T-DNA integration occurred in these transformants. The rearrangements were further con W rmed by sequencing the host–host rejoining sites for each transformant. Taking these results together, translocation occurred in transformant 438, with large fragments translocated between chromosomes I and IV (Fig.4d), while inversions occurred in transformant 6001 and 4825, involving fragments with 64 and 218kb in size, respectively (Fig.4e).Southern blot analysis of transformant 6001 using six probes derived from representative locations of chromosomal fragments involved in arrangement, further con W rmed the inversion event (Fig.5). Fig.5IIa, e, f indicated that no duplication presented in the T-DNA X anking regions since only one band was observed for 6001 consistently. Fig.5IIc, d showed that genomic DNAs between the two pre-dicted T-DNA insertion sites (Fig.5I) remained intact. Fig.5IIb indicated that there was another rejoining site (host–host) existed somewhere in the genome of 6001. Therefore, together with evidences of PCR analysis and host–host junction sequencing mentioned above, chromosomal inversion event was con W rmed further in transformant 6001.Analysis of sequences of rearrangemental junction regions (including host-LB, RB-host, and host–host) revealed that rejoining patterns similar to ones described above for general T-DNA integration (Tables3, 4) were observed. Small fragment deletions and microhomologies were found frequently. Dele-tions of host genomic sequences with 30, 12, and 22bp in size were observed in transformant 6001, 4825, and 438, respectively. Microhomologies with 1–3bp were found in 5 of 9 rearrangement sites. No W ller sequence was observed at these junction sites in all three tested transformants.Table3Sequence analysis of junctions between T-DNA borders and genomic target sitesRB LBSize (bp)Number of junctions Size (bp)Number of junctions Identical nucleotides between target site and border1–5971–7901701122–5272–778Filler nucleotides between target site and border1–230561–221–303330–23023Exact joining between target site and border30932Total sequences analyzed462124Table4Sequences analysis of T-DNA insertion sitesNumber of T-DNAinsertionsDuplication of target site1–8bp 5 (6.7%)Exact integration into target site 6 (8.0%)Deletion of target site61 (81.3%)1–20bp46 (61.3%)21–80bp14 (18.7%)>80bp 1 (1.3%)Inversions 2 (2.7%)Translocations 1 (1.3%)Number of sequences analyzed75(100%)Complex T-DNA integration patternsOf the 623 RB X anking sequences, 65 (10.4%) were involved in long T-DNA integration. Southern blot analysis (data not shown) with probes derived from di V erent regions of pBHt2 suggested that some trans-formants might contain at least one copy of the whole binary vector. To test that speculation, W ve transfor-mants were selected for further assay of plasmid rescue. Genomic DNAs were digested with Pst I and self-ligated. Plasmids were rescued from four transfor-mants but failed from one. Analysis of restriction endonucleases digestions and PCR reactions indicated that these retrieved plasmids were the same as the orig-inal plasmid pBHt2 (data not shown), suggesting that long T-DNA with greater-than-unit-length of the binary plasmid has been integrated into the genome of M. oryzae .Analysis of RB X anking sequences also revealed that 11.4% (71 of 623) of transformants contained two T-DNAs (one might be partial) co-integrated into the genome of M. oryzae with head-to-tail fusions (direct repeat). At the junctions, rejoining patterns similar to ones described above for general T-DNA integration (Fig.3, Table 3) were also observed. Most RBs (98.6%) showed full-length fusions, but one had 1bp deletion. Sometimes, LBs undergone deletions of 1–180bp, and full-length LB fusions were found in 44 (62.0%) transformants.Filler sequence (4.2%, with 1bp) and microhomology (2.8%, with 1–2bp) were also found but rarely at the junctions.Fig.4PCR analysis and pro-posed modes of chromosomal rearrangements. Each puta-tive transformant, 6001(a ), 4825 (b ) or 438 (c ) were analyzed by PCR using four sets of primer pairs (which relative positions were shown in d or e ) as indicated on the top of each panel. Wild types Y34 and 70-15 were used as control. Original strains of templates were shown under primers. T1, T2, and T3 de-note transformant 6001, 4825, and 438, respectively; W1 and W2 denote Y34 and 70-15, respectively; M, 1kb DNA marker. Modes of chromo-somal rearrangement associ-ated with T-DNA integration are proposed based on molec-ular evidences of this study. When invading T-DNA mole-cule is physically close to host chromatins where two DSB events existed simultaneously, rearrangement might happen. d Translocation occurs (as transformant 438) if the two DSB events involved in rear-rangement derived from di V erent chromosomes. e Inversion occurs (as trans-formant 6001 and 4825) if the two involved DSB events originated from a same chromosome. DSB , double-stranded break。