NBS-LRR resistance gene homologues in rice

Twenty three DNA fragments with a size of about 520 bp have been cloned from rice genome by PCR amplification using primers designed according to the conserved region of most plant resistance (R) genes which have Nucleotide Binding Site (NBS) and Leucine-Rich Repeat (LRR) domains. Homologous comparison showed that these fragments contained typical motifs of the NBS-LRR resistance gene class, kinase 1a, kinase 2a, kinase 3a and domain 2. Thus they were named R gene homologous sequences (RS). These RS were divided into 4 groups by clustering analysis and mapped onto chromosomes 1, 3, 4, 7, 8, 9, 10 and 11, respectively, by genetic mapping. Ten RS were located in the chromosomal intervals where known R genes had been mapped. Further RFLP analysis of an RS, RS13, near the bacterial blight resistance gene Xa4 locus on chromosome 11 among near isogenic lines and pyramiding lines of Xa4 showed that RS13 was possibly amplified from the gene family of Xa4.     

Evaluation of resistance genes in rice against local isolates of Xanthomonas oryzae pv. oryzae in Punjab Province of Pakistan

Absence of resistance/tolerance against bacterial leaf blight (BLB), incited by Xanthomonas oryzae pv. oryzae, in famous basmati varieties is one of the main reason for BLB epidemic in Punjab in 2007–2008. For developing resistance against BLB, the response of 26 IRBB lines of IRRI including 10 near isogenic lines (NILs) and 16 gene pyramids carrying two to five resistance genes (Xa series) was evaluated against 61 indigenous Xoo isolates under artificial inoculation field conditions. None of the NILs or gene pyramid provides complete protection against all the isolates. However, Xa21 and xa13 were found resistant against the majority of Xoo isolates, followed by Xa14 and Xa7. Of the 16 gene pyramids used in this study, IRBB-54 (Xa5 + Xa21), IRBB-55 (Xa13 + Xa21) followed by IRBB-58 (Xa4 + Xa13 + Xa21) were found effective against the majority of the Xoo isolates. These resistance genes (individually and in combinations) can be incorporated for the improvement of basmati rice cultivars cultivated in Punjab province of Pakistan. Effectiveness of gene combination supports the strategy of pyramiding appropriate resistance genes. Newly identified resistant genes may also be evaluated for achieving broad spectrum resistance against more Xoo isolates of the area.     

RFLP mapping and race specificity of bacterial blight resistance genes (QTLs) in rice

ＢａｃｔｅｒｉａｌｂｌｉｇｈｔｃａｕｓｅｄｂｙＸａｎｔｈｏｍｏｎａｓｏｒｙｚａｅｐｖ.ｏｒｙｚａ(Ｘｏｏ)ｉｓｏｎｅｏｆｔｈｅｍｏｓｔｓｅｒｉｏｕｓｒｉｃｅｄｉｓｅａｓｅｓｉｎＡｓｉａ.Ｓｉｎｃｅｔｈｅ1920ｓ,ｐｒｏｂｉｎｇｔｈｅｇｅｎｅｔｉｃｂａｓｉｓｏｆｒｅｓｉｓｔａｎｃｅｔｏＸｏｏａｎｄｂｒｅｅｄｉｎｇｒｅｓｉｓｔａｎｔｖａｒｉｅｔｉｅｓｈａｖｅｂｅｅｎａｈｏｔｐｏｉｎｔｏｆｒｉｃｅｓｃｉｅｎｃｅｒｅｓｅａｒｃｈ[1,2].Ｂｏｔｈｔｙｐｅｓｏｆｒｅｓｉｓｔａｎｃｅ,ＶＲ(ｑｕａｌｉｔａｔｉｖｅｒｅｓ…     

Marker assisted selection of bacterial blight resistance genes in rice

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae is one of the most important diseases affecting rice production in Asia. We were interested in surveying rice genotypes that are popularly used in the Indian breeding program for conferring resistance to bacterial blight, using 11 STMS and 6 STS markers. The basis of selection of these DNA markers was their close linkage to xa5, xa13, and Xa21 genes and their positions on the rice genetic map relative to bacterial blight resistance genes. Eight lines were found to contain the xa5 gene while two lines contained Xa21 gene and none of the lines contained the xa13 gene with the exception of its near-isogenic line. Using the polymorphic markers obtained in the initial survey, marker-assisted selection was performed in the F3 population of a cross between IR-64 and IET-14444 to detect lines containing multiple resistance genes. Of the 59 progeny lines analyzed, eight lines contained both the resistance genes, xa5 and Xa4.     

Cloning and characterization of receptor kinase class disease resistance gene candidates in Citrus

The rice gene Xa21 represents a unique class of plant disease resistance (R) genes with distinct protein structure and broad-spectrum specificity; few sequences or genes of this class have been cloned and characterized in other plant species. Degenerate primers were designed from the conserved motifs in the kinase domains of Xa21 and tomato Pto, and used in PCR amplification to identify this class of resistance gene candidate (RGC) sequences from citrus for future evaluation of possible association with citrus canker resistance. Twenty-nine RGC sequences highly similar to the kinase domain of Xa21 (55%–60% amino-acid identity) were cloned and characterized. To facilitate recovery of full-length gene structures and to overcome RGC mapping limitations, large-insert genomic clones (BACs) were identified, fingerprinted and assembled into contigs. Southern hybridization revealed the presence of 1–3 copies of receptor-like kinase sequences (i.e., clustering) in each BAC. Some of these sequences were sampled by PCR amplification and direct sequencing. Twenty-three sequences were thus obtained and classified into five groups and eight subgroups, which indicates the possibility of enhancing RGC sequence diversity from BACs. A primer-walking strategy was employed to derive full-length gene structures from two BAC clones; both sequences 17o6RLK and 26m19RLK contained all the features of the rice Xa21 protein, including a signal peptide, the same number of leucine-rich-repeats, and transmembrane and kinase domains. These results demonstrate that PCR amplification with appropriately designed degenerate primers is an efficient approach for cloning receptor-like kinase class RGCs. Utilization of BAC clones can facilitate this approach in multiple ways by improving sequence diversity, providing full-length genes, and assisting in understanding gene structures and distribution.Communicated by P. Langridge     

A "defeated" rice resistance gene acts as a QTL against a virulent strain of Xanthomonas oryzae pv. oryzae

The genetic components responsible for qualitative and quantitative resistance of rice plants to three strains (CR4, CXO8, and CR6) of Xanthomonas oryzae pv. oryzae (Xoo) were investigated using a set of 315 recombinant inbred lines (RILs) from the cross Lemont (japonica) × Teqing (indica) and a complete linkage map with 182 well distributed RFLP markers. We mapped a major gene (Xa4) and ten quantitative trait loci (QTLs) which were largely responsible for segregation of the resistance phenotype in the RILs. The Teqing allele at the Xa4 locus, Xa4 ^T , acted as a dominant resistance gene against CR4 and CXO8. The breakdown of Xa4 ^T -associated resistance mediated by the mutant allele at the avrXa4 locus in the virulent strain CR6 results from significant changes in both gene action (lose of dominance) and the magnitude of gene effect (≈50% reduction). Nevertheless, Xa4 ^T still acted as a recessive QTL with a significant residual effect against CR6. The mutant alleles at the avrXa4 locus in CXO8 and CR6 that lead to a reduction in effect, or “breakdown”, of Xa4 ^T were apparently accompanied by corresponding penalties for their fitness. The quantitative component of resistance to Xoo in the RILs was largely due to a number of resistance QTLs. Most resistance QTLs mapped to genomic locations where major resistance genes and/or QTLs for resistance to Xoo, blast and sheath blight were identified in the same cross. Most QTLs showed consistent levels of resistance against all three Xoo strains. Our results suggest that a high level of durable resistance to Xoo may be achieved by the cumulative effects of multiple QTLs, including the residual effects of “defeated” major resistance genes. Received: 28 April 1998 / Accepted: 9 October 1998     

水稻抗白叶枯病基因Xa4位点跨叠BAC克隆群的构建

水稻白叶枯病抗性基因Xa4已被定位于第11染色体长臂末端的分子标记VG181和L1044之间,并与抗性基因同源序列片段RS13共分离。利用这3个标记筛选IRBB56的BAC文库,共得到128个阳性BAC克隆,其中RS13获得18个阳性克隆,这18个克隆中有4个和6个我隆分别同时为G181和L1044的阳性克隆,选其中的12克隆进行分析,构建了一个从G181到L1044区间的BAC跨叠克隆,全长420kb,并且56M22、106P13和104B153个BAC克隆可覆盖整个跨叠克隆群。这一研究结果为进一步分离Xa4基因打下基础。     

High-resolution mapping and gene prediction of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> resistance gene <Emphasis Type="Italic">Xa7</Emphasis>

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease in rice worldwide. The resistance gene Xa7, which provides dominant resistance against the pathogen with avirulence (Avr) gene AvrXa7, has proved to be durably resistant to BB. A set of SSR markers were selected from the “gramene” database based on the Xa7 gene initial mapping region on chromosome 6. These markers were used to construct a high-resolution genetic map of the chromosomal region surrounding the Xa7 gene. An F₂ mapping population with 721 highly susceptible individuals derived from a cross between the near isogenic lines (NILs) IRBB7 and IR24 were constructed to localize the Xa7 gene. In a primary analysis with eleven polymorphic SSR markers, Xa7 was located in approximately the 0.28-cM region. To walk closer to the target gene, recombinant F₂ individuals were tested using newly developed STMS (sequence tagged microsatellite) markers. Finally, the Xa7 gene was mapped to a 0.21-cM interval between the markers GDSSR02 and RM20593. The Xa7-linked markers were landed on the reference sequence of cv. Nipponbare through bioinformatics analysis. A contig map corresponding to the Xa7 gene was constructed. The target gene was assumed to span an interval of approximately 118.5-kb which contained a total of fourteen genes released by the TIGR Genome Annotation Version 5.0. Candidate-gene analysis of Xa7 revealed that the fourteen genes encode novel domains that have no amino acid sequence similar to other cloned Xa(xa) genes. Shen Chen and Zhanghui Huang are contributed equally to this work.     

Genotypic and Pathotypic Diversity of Xanthomonas oryzae pv. oryzae Strains in Taiwan

Rice bacterial leaf blight, caused by Xanthomonas oryzae pv. oryzae [(Ishiyama) Swings et al. 1990] (Xoo), is a major rice disease of the second crop season in Taiwan. A total of 88 Xoo strains collected from 10 major rice cultivating areas in Taiwan from 1986, 1997, 2000, 2004, and 2011 were characterized by repetitive‐element PCR (REP‐PCR) fingerprinting and virulence analyses. Among the five genetic clusters identified by the pJEL1/pJEL2 (IS1112‐based) and REP1R‐Dt/REP2‐D [repetitive extragenic palindromic (REP)‐based] primer sets, clusters A, C and D contained Xoo strains from geographically distant regions, which suggests a high frequency of Xoo dispersal in Taiwan. The 88 Xoo strains were evaluated by inoculations on IRBB near‐isogenic lines and five Taiwan rice cultivars. A subset of 45 moderately or highly virulent strains were classified into 15 pathotypes by their compatible or incompatible reactions on IR24 and 12 IRBB near‐isogenic lines, each containing a single resistance gene. Analysis of molecular haplotypes and pathotypes revealed a partial relationship. IRBB5, IRBB21 and IRBB4 were incompatible with 96%, 96% and 73% of the strains, so xa5, Xa21 and Xa4 can recognize most of the Xoo strains in Taiwan and elicit resistance. In contrast, IRBB3 (Xa3), IRBB8 (xa8), IRBB10 (Xa10), IRBB11 (Xa11), IRBB13 (xa13) and IRBB14 (Xa14) were susceptible to almost all of the 45 Xoo strains. Inoculation trials revealed significant differences in the susceptibility of five Taiwan cultivars to Xoo (from high to low susceptibility: Taichung Sen 10 >  IR24, Taichung Native 1 >  Taichung 192, Taikeng 9, Tainan 11). This study provides useful information for resistance breeding and the development of disease management strategies against bacterial blight disease of rice.     

Pyramiding transgenic resistance in elite indica rice cultivars against the sheath blight and bacterial blight

Elite indica rice cultivars were cotransformed with genes expressing a rice chitinase (chi11) and a thaumatin-like protein (tlp) conferring resistance to fungal pathogens and a serine-threonine kinase (Xa21) conferring bacterial blight resistance, through particle bombardment, with a view to pyramiding sheath blight and bacterial blight resistance. Molecular analyses of putative transgenic lines by polymerase chain reaction, Southern Blot hybridization, and Western Blotting revealed stable integration and expression of the transgenes in a few independent transgenic lines. Progeny analyses showed the stable inheritance of transgenes to their progeny. Coexpression of chitinase and thaumatin-like protein in the progenies of a transgenic Pusa Basmati1 line revealed an enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, as compared to that in the lines expressing the individual genes. A transgenic Pusa Basmati1 line pyramided with chi11, tlp, and Xa21 showed an enhanced resistance to both sheath blight and bacterial blight. S. Maruthasalam and K. Kalpana have contributed to this article equally.     

Cloning and in silico Mapping of Resistance Gene Analogues Isolated from Rice Lines Containing Known Genes for Blast Resistance

We amplified resistance gene analogues (RGAs) from the genomic DNA of 10 rice lines having varying degree of resistance to Magnaporthe grisea by using degenerate primers and various RGAs were mapped in silico on different rice chromosomes. The amplified products were grouped into 3–8 restriction fragment length polymorphic classes by using Mbo1 and Alu1 restriction enzymes. Of 98 RGAs obtained in this study, 65 RGA clones showed more than 95% homology with various RGAs sequences present in the GenBank. Phylogenetic analysis of these RGAs formed 11 groups. Using sequence homology approach, RGAs isolated in this study were physically mapped on 23 loci on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 10, 11 and 12. Twenty RGAs were mapped near to the chromosomal regions containing known genes/QTLs for rice blast, bacterial leaf blight and sheath blight resistance. Thirty‐nine RGA sequences also contained open reading frame representing signature of potential disease resistance genes.     

High-resolution genetic mapping of rice bacterial blight resistance gene Xa23

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.     

Isolation and characterization of rice mutants compromised in<Emphasis Type="Italic"> Xa21</Emphasis>-mediated resistance to<Emphasis Type="Italic"> X. oryzae</Emphasis> pv.<Emphasis Type="Italic"> oryzae</Emphasis>

The rice gene, Xa21, confers resistance to diverse races of Xanthomonas oryzae pv. oryzae (Xoo) and encodes a receptor-like kinase with leucine-rich repeats in the extra-cellular domain. To identify genes essential for the function of the Xa21 gene, 4,500 IRBB21 (Xa21 isogenic line in IR24 background) mutants, induced by diepoxybutane and fast neutrons, were screened against Philippine race six (PR6) Xoo for a change from resistance to susceptibility. From two greenhouse screens, 23 mutants were identified that had changed from resistant to fully (6) or partially (17) susceptible to PR6. All fully susceptible mutants carried rearrangements at the Xa21 locus as detected by PCR and Southern hybridization. For the partially susceptible mutants, no changes were detected at the Xa21 locus based on Southern and PCR analyses. However, two of these mutants were confirmed via genetic analysis to have mutations at the Xa21 locus. Partially susceptible mutants exhibited variation in level of susceptibility to different Xoo strains, suggesting that they may carry different mutations required for the Xa21-mediated resistance. The mutants identified in this study provide useful materials for dissecting the Xa21-mediated resistance pathway in rice.Communicated by D.J. Mackill     

Identification and fine mapping of a resistance gene to <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in a space-induced rice mutant

Finding novel sources of resistance (R) to rice blast disease should facilitate breeding for improved resistance. The objectives of the present study were to evaluate reactions to blast and identify in a space-induced mutant an R gene to a representative isolate of rice blast pathogen. The mutant H4, its parent and twelve monogenic lines were evaluated for their responses to 35 isolates collected from Guangdong Province, China. H4 was found to be resistant to more isolates than its parent and the twelve monogenic lines, suggesting newly acquired resistance may be a function of one or more R genes. A representative isolate GD0193 was used to identify and map the R gene from H4. Genetic analysis revealed that resistance to the isolate GD0193 was controlled by a single dominant gene, designated Pi46(t). Linkage analysis using susceptible F2 individuals showed that Pi46(t) was mapped between the markers RM224 and RM27360 within 1.04 and 1.2 cM on the long arm of chromosome 11. Subsequently, Pi46(t) was delimited to an interval of approximately 183.7 kb flanked by the markers K67 and T94. These results provide essential information for the cloning of the Pi46(t) gene and will facilitate marker-assisted selection in rice breeding.     

Linkage mapping of genes for resistance to leaf,stem and stripe rusts and ω-secalins on the short arm of rye chromosome 1R

Summary The genes controlling resistance to three wheat rusts, viz., leaf rust (Lr26), stem rust (Sr31) and stripe or yellow rust (Yr9), and -secalins (Sec1), located on the short arm of rye chromosome 1R, were mapped with respect to each other and the centromere. Analysis of 214 seeds (or families derived from them) from testcrosses between a 1BL.1RS/1R heterozygote and Chinese Spring ditelocentric 1BL showed no recombination between the genes for resistance to the three rusts, suggesting very tight linkage or perhaps a single complex locus conferring resistance to the three rusts. The rust resistance genes were located 5.4 ± 1.7 cM from the Sec1 locus, which in turn was located 26.1 ± 4.3 cM from the centromere; the gene order being centromere — Sec1 — Lr26/Sr31/Yr9 — telomere. In a second test-cross, using a different 1BL.1RS translocation which had only stem rust resistance (SrR), the above gene order was confirmed despite a very large proportion of aneuploids (45.8%) among the progeny. Furthermore, a map distance of 16.0 ± 4.8 cM was estimated for SrR and the telomeric heterochromatin (C-band) on 1RS. These results suggest that a very small segment of 1RS chromatin is required to maintain resistance to all three wheat rusts. It should be possible but difficult to separate the rust resistance genes from the secalin gene(s), which are thought to contribute to dough stickiness of wheat-rye translocation lines carrying 1RS.     

Resistance gene analogues from rice: cloning, sequencing and mapping

 Degenerate oligonucleotide primers were designed on the basis of nucleotide-binding-site (NBS) motifs conserved between resistance genes of Arabidopsis, flax and tobacco and subsequently used as PCR primers to amplify resistance gene analogues (RGA) in rice. Primers amplified a major band of approximately 500 bp. Restriction analysis of the amplified product revealed that the band was made up of several different fragments. Many of these fragments were cloned. Sixty different cloned fragments were analysed and assigned to 14 categories based on Southern blot analysis. Fourteen clones, each representing one of the 14 categories of RGAs were mapped onto the rice genetic map using a Nipponbare ( japonica)בKasalath’ (indica) mapping population consisting of 186 F₂ lines. Of the 14 clones representing each class 12 could be mapped onto five different chromosomes of rice with a major cluster of 8 RGAs on chromosome 11. Our results indicate that it is possible to use sequence homology from conserved motifs of known resistance genes to amplify candidate resistance genes from diverse plant taxa. Received: 23 September 1998 / Accepted: 28 November 1998     

Identification and mapping of molecular markers linked to rust resistance genes located on chromosome 1RS of rye using wheat-rye translocation lines

The short arm of rye (Secale cereale) chromosome 1 has been widely used in breeding programs to incorporate new disease resistance genes into wheat. Using wheat-rye translocation and recombinant lines, molecular markers were isolated and mapped within chromosomal regions of 1RS carrying rust resistance genes Lr26, Sr31, Yr9 from 'Petkus' and SrR from 'Imperial' rye. RFLP markers previously mapped to chromosome 1HS of barley - flanking the complex Mla powdery mildew resistance gene locus - and chromosome 1DS of Aegilops tauschii - flanking the Sr33 stem rust resistance gene - were shown to map on either side of rust resistance genes on 1RS. Three non cross-hybridising Resistance Gene Analog markers, one of them being derived from the Mla gene family, were mapped within same region of 1RS. PCR-based markers were developed which were tightly linked to the rust resistance genes in 'Imperial' and 'Petkus' rye and which have potential for use in marker-assisted breeding.     

Xa3, conferring resistance for rice bacterial blight and encoding a receptor kinase-like protein, is the same as Xa26

Xa3-mediated resistance for rice bacterial blight, one of the most devastating rice diseases worldwide, is influenced by genetic background. Xa3 is genetically tightly linked to Xa26, another gene for bacterial blight resistance. Xa26 belongs to a clustered multigene family encoding leucine-rich repeat (LRR) receptor kinase-like proteins. To characterize Xa3, we fine mapped it using a population segregating for only one resistance gene and markers developed from Xa26 family. Genetic analysis showed that Xa3 co-segregated with the marker of Xa26 gene and segregated from the markers of other members of Xa26 family. DNA fingerprinting revealed that rice line IRBB3 carrying Xa3 had the same copy numbers of Xa26 family members as rice line Minghui 63 carrying Xa26. Phenotypic comparison showed that all the rice lines carrying either Xa3 or Xa26 developed dark brown deposition at the border between the lesion caused by incompatible-pathogen infection and health leaf tissue, while other rice lines did not show this dark brown deposition in either incompatible or compatible interactions. These results suggest that Xa3 and Xa26 is the same gene. We name it Xa3/Xa26 to indicate the relationship between the two gene symbols. The putative encoding products of Xa3/Xa26 and its susceptible allele xa3/xa26 shared 92% sequence identity. The sequence difference occurred in the LRR domains, specifically at the solvent-exposed amino acid residues, might be the major cause that differentiates the resistant and susceptible proteins.     

Identification of the novel recessive gene pi55(t) conferring resistance to Magnaporthe oryzae

The elite rice cultivar Yuejingsimiao 2 (YJ2) is characterized by a high level of grain quality and yield, and resistance against Magnaporthe oryzae. YJ2 showed 100% resistance to four fungal populations collected from Guangdong, Sichuan, Liaoning, and Heilongjiang Provinces, which is a higher frequency than that shown by the well-known resistance (R) gene donor cultivars such as Sanhuangzhan 2 and 28zhan. Segregation analysis for resistance with F₂ and F₄ populations indicated the resistance of YJ2 was controlled by multiple genes that are dominant or recessive. The putative R genes of YJ2 were roughly tagged by SSR markers, located on chromosomes 2, 6, 8, and 12, in a bulked-segregant analysis using genome-wide selected SSR markers with F₄ lines that segregated into 3 resistant (R):1 susceptible (S) or 1R:3S. The recessive R gene on chromosome 8 was further mapped to an interval ≈1.9 cM/152 kb in length by linkage analysis with genomic position-ready markers in the mapping population derived from an F₄ line that segregated into 1R:3S. Given that no major R gene was mapped to this interval, the novel R gene was designated as pi55(t). Out of 26 candidate genes predicted in the region based on the reference genomic sequence of the cultivar Nipponbare, two genes that encode a leucine-rich repeat-containing protein and heavy-metal-associated domain-containing protein, respectively, were suggested as the most likely candidates for pi55(t).