Molecular characterization of a wheat–Psathyrostachys huashanica Keng 2Ns disomic addition line with resistance to stripe rust

We characterized a wheat–Psathyrostachys huashanica derived line 3-6-4-1 based on genomic in situ hybridization (GISH), molecular marker analysis, and agronomic trait evaluations. The GISH investigations showed that the 3-6-4-1 contained 42 wheat chromosomes and a pair of P. huashanica chromosomes. The homoeologous relationships of the introduced P. huashanica chromosomes were determined using EST-STS multiple loci markers from seven wheat homoeologous groups in the parents and the addition line. Twelve EST-STS markers located on the homoeologous group 2 chromosomes of wheat amplified polymorphic bands in 3-6-4-1, which were unique to P. huashanica. An introduced Ns chromosome pair that belonged to homoeologous group 2 was identified using chromosome-specific markers. Inoculation with isolates of the stripe rust pathotypes, CYR31, CYR32, and SY11-14, and mixed races (CYR31, CYR32, and SY11-14) in the seeding and adult stage, respectively, showed that 3-6-4-1 was generally resistant to stripe rust, which was probably attributable to its P. huashanica parent. We also compared a complete set of wheat–P. huashanica disomic addition lines (1Ns–7Ns, 2n = 44 = 22II) to assess their agronomic traits and morphological characteristics, which showed that 3-6-4-1 had improved spike traits compared with its parents. The P. huashanica 2Ns chromosome-specific molecular markers in 3-6-4-1 could be useful for marker-assisted selection in breeding programs to combat stripe rust. This line can be used as a donor source to introduce novel excellent genes from P. huashanica into wheat to widen its genetic diversity, thereby providing new germplasms for wheat breeding.     

TaLHY,a 1R-MYB Transcription Factor,Plays an Important Role in Disease Resistance against Stripe Rust Fungus and Ear Heading in Wheat

LHY (late elongated hypocotyl) is an important gene that regulates and controls biological rhythms in plants. Additionally, LHY is highly expressed in the SSH (suppression subtractive hybridization) cDNA library-induced stripe rust pathogen (CYR32) in our previous research. To identify the function of the LHY gene in disease resistance against stripe rust, we used RACE-PCR technology to clone TaLHY in the wheat variety Chuannong19. The cDNA of TaLHY is 3085 bp long with an open reading frame of 1947 bp. TaLHY is speculated to encode a 70.3 kDa protein of 648 amino acids , which has one typical plant MYB-DNA binding domain; additionally, phylogenetic tree shows that TaLHY has the highest homology with LHY of Brachypodium distachyon(BdLHY-like). Quantitative fluorescence PCR indicates that TaLHY has higher expression in the leaf, ear and stem of wheat but lower expression in the root. Infestation of CYR32 can result in up-regulated expression of TaLHY, peaking at 72 h. Using VIGS (virus-induced gene silencing) technology to disease-resistant wheat in the fourth leaf stage, plants with silenced TaLHY cannot complete their heading stage. Through the compatible interaction with the stripe rust physiological race CYR32, Chuannong 19 loses its immune capability toward the stripe rust pathogen, indicating that TaLHY may regulate and participate in the heading of wheat, as well as the defense responses against stripe rust infection.     

Development of a wheat single gene FISH map for analyzing homoeologous relationship and chromosomal rearrangements within the Triticeae

Key message

A cytogenetic map of wheat was constructed using FISH with cDNA probes. FISH markers detected homoeology and chromosomal rearrangements of wild relatives, an important source of genes for wheat improvement.

Abstract

To transfer agronomically important genes from wild relatives to bread wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) by induced homoeologous recombination, it is important to know the chromosomal relationships of the species involved. Fluorescence in situ hybridization (FISH) can be used to study chromosome structure. The genomes of allohexaploid bread wheat and other species from the Triticeae tribe are colinear to some extent, i.e., composed of homoeoloci at similar positions along the chromosomes, and with genic regions being highly conserved. To develop cytogenetic markers specific for genic regions of wheat homoeologs, we selected more than 60 full-length wheat cDNAs using BLAST against mapped expressed sequence tags and used them as FISH probes. Most probes produced signals on all three homoeologous chromosomes at the expected positions. We developed a wheat physical map with several cDNA markers located on each of the 14 homoeologous chromosome arms. The FISH markers confirmed chromosome rearrangements within wheat genomes and were successfully used to study chromosome structure and homoeology in wild Triticeae species. FISH analysis detected 1U-6U chromosome translocation in the genome of Aegilops umbellulata, showed colinearity between chromosome A of Ae. caudata and group-1 wheat chromosomes, and between chromosome arm 7S#3L of Thinopyrum intermedium and the long arm of the group-7 wheat chromosomes.     

Genetic mapping of a putative Thinopyrum intermedium-derived stripe rust resistance gene on wheat chromosome 1B

Key message

Stripe rust resistance transferred from Thinopyrum intermedium into common wheat was controlled by a single dominant gene, which mapped to chromosome 1B near Yr26 and was designated YrL693.

Abstract

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a highly destructive disease of wheat (Triticum aestivum). Stripe rust resistance was transferred from Thinopyrum intermedium to common wheat, and the resulting introgression line (L693) exhibited all-stage resistance to the widely virulent and predominant Chinese pathotypes CYR32 and CYR33 and to the new virulent pathotype V26. There was no cytological evidence that L693 had alien chromosomal segments from Th. intermedium. Genetic analysis of stripe rust resistance was performed by crossing L693 with the susceptible line L661. F₁, F₂, and F_2:3 populations from reciprocal crosses showed that resistance was controlled by a single dominant gene. A total 479 F_2:3 lines and 781 pairs of genomic simple sequence repeat (SSR) primers were employed to determine the chromosomal location of the resistance gene. The gene was linked to six publicly available and three recently developed wheat genomic SSR markers. The linked markers were localized to wheat chromosome 1B using Chinese Spring nulli-tetrasomic lines, and the resistance gene was localized to chromosome 1B based on SSR and wheat genomic information. A high-density genetic map was also produced. The pedigree, molecular marker data, and resistance response indicated that the stripe rust resistance gene in L693 is a novel gene, which was temporarily designated YrL693. The SSR markers that co-segregate with this gene (Xbarc187-1B, Xbarc187-1B-1, Xgwm18-1B, and Xgwm11-1B) have potential application in marker-assisted breeding of wheat, and YrL693 will be useful for broadening the genetic basis of stripe rust resistance in wheat.     

Molecular cytogenetic identification of a wheat–Psathyrostachys huashanica Keng 5Ns disomic addition line with stripe rust resistance

We developed a new stripe rust resistant line of common wheat–Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) from a cross between wheat cv. 7182 and P. huashanica via embryo culture, and we refer to this line as 3-8-10-2. We characterized this new line by cytology, genomic in situ hybridization (GISH), EST-SSR, EST-STS, and disease resistance screening. GISH using P. huashanica genomic DNA as the probe indicated that a pair of Ns chromosomes with strong hybridization signals was introduced into 3-8-10-2. We screened 255 EST-SSR and EST-STS multiple-loci markers from seven wheat homoeologous groups in the parent lines. Of these, 90 markers were polymorphic with a polymorphism frequency of 40 %, while two EST-SSR markers and six EST-STS markers located on wheat chromosome group 5 produced specific bands in P. huashanica and 3-8-10-2, respectively. This suggested that the introduced Ns chromosome pair belonged to homoeologous group 5, which was identified using new genome-specific markers. After inoculation with stripe rust isolates, 3-8-10-2 exhibited stripe rust resistance that probably originated from its P. huashanica parent. 3-8-10-2 can be used as a donor source for introducing novel disease resistance genes into wheat during breeding programs with the assistance of molecular and cytogenetic markers. Moreover, 3-8-10-2 had improved agronomic characteristics compared with its parents. Therefore, the addition line could be exploited as an important bridge for wheat breeding and chromosome engineering.     

Nostoc, Microcoleus and Leptolyngbya inoculums are detrimental to the growth of wheat (Triticum aestivum L.) under salt stress

Background and aims

This study investigated the effect of cyanobacterial inoculants on salt tolerance in wheat.

Methods

Unicyanobacterial crusts of Nostoc, Leptolyngbya and Microcoleus were established in sand pots. Salt stress was targeted at 6 and 13 dS m^?1, corresponding to the wheat salt tolerance and 50 % yield reduction thresholds, respectively. Germinated wheat seeds were planted and grown for 14 (0 and 6 dS m^?1) and 21 (13 dS m^?1) days by which time seedlings had five emergent leaves. The effects of cyanobacterial inoculation and salinity on wheat growth were quantified using chlorophyll fluorescence, inductively coupled plasma-optical emission spectrometry and biomass measurements.

Results

Chlorophyll fluorescence was negatively affected by soil salinity and no change was observed in inoculated wheat. Effective photochemical efficiency correlated with a large range of plant nutrient concentrations primarily in plant roots. Inoculation negatively affected wheat biomass and nutrient concentrations at all salinities, though the effects were fewer as salinity increased.

Conclusions

The most likely explanation of these results is the sorption of nutrients to cyanobacterial extracellular polymeric substances, making them unavailable for plant uptake. These results suggest that cyanobacterial inoculation may not be appropriate for establishing wheat in saline soils but that cyanobacteria could be very useful for stabilising soils.     

Mapping stripe rust resistance gene <Emphasis Type="Italic">YrZH22</Emphasis> in Chinese wheat cultivar Zhoumai 22 by bulked segregant RNA-Seq (BSR-Seq) and comparative genomics analyses

Key message

A stripe rust resistance gene YrZH22 was mapped by combined BSR-Seq and comparative genomics analyses to a 5.92 centimorgan (cM) genetic interval spanning a 4 Mb physical genomic region on wheat chromosome 4BL1.

Abstract

Stripe rust, caused by Puccinia striiformis f. sp. tritici (PST), is one of the most destructive diseases of wheat and severely threatens wheat production worldwide. The widely grown Chinese wheat cultivar Zhoumai 22 is highly resistant to the current prevailing PST race CYR34 (V26). Genetic analysis of F_5:6 and F_6:7 recombinant inbred line (RIL) populations indicated that adult-plant stripe rust resistance in Zhoumai 22 is controlled by a single gene, temporarily designated YrZH22. By applying bulked segregant RNA-Seq (BSR-Seq), 7 SNP markers were developed and SNP mapping showed that YrZH22 is located between markers WGGB105 and WGGB112 on chromosome arm 4BL. The corresponding genomic regions of the Chinese Spring 4BL genome assembly and physical map of Aegilops tauschii 4DL were selected for comparative genomics analyses to develop nine new polymorphic markers that were used to construct a high-resolution genetic linkage map of YrZH22. YrZH22 was delimited in a 5.92 cM genetic interval between markers WGGB133 and WGGB146, corresponding to 4.1 Mb genomic interval in Chinese Spring 4BL and a 2.2 Mb orthologous genomic region in Ae. tauschii 4DL. The genetic linkage map of YrZH22 will be valuable for fine mapping and positional cloning of YrZH22, and can be used for marker-assisted selection in wheat breeding.

     

Evaluation of reference genes for real-time RT-PCR expression studies in the plant pathogen Pectobacterium atrosepticum

Background

Translation initiation factors of the 4E and 4G protein families mediate resistance to several RNA plant viruses in the natural diversity of crops. Particularly, a single point mutation in melon eukaryotic translation initiation factor 4E (eIF4E) controls resistance to Melon necrotic spot virus (MNSV) in melon. Identification of allelic variants within natural populations by EcoTILLING has become a rapid genotype discovery method.

Results

A collection of Cucumis spp. was characterised for susceptibility to MNSV and Cucumber vein yellowing virus (CVYV) and used for the implementation of EcoTILLING to identify new allelic variants of eIF4E. A high conservation of eIF4E exonic regions was found, with six polymorphic sites identified out of EcoTILLING 113 accessions. Sequencing of regions surrounding polymorphisms revealed that all of them corresponded to silent nucleotide changes and just one to a non-silent change correlating with MNSV resistance. Except for the MNSV case, no correlation was found between variation of eIF4E and virus resistance, suggesting the implication of different and/or additional genes in previously identified resistance phenotypes. We have also characterized a new allele of eIF4E from Cucumis zeyheri, a wild relative of melon. Functional analyses suggested that this new eIF4E allele might be responsible for resistance to MNSV.

Conclusion

This study shows the applicability of EcoTILLING in Cucumis spp., but given the conservation of eIF4E, new candidate genes should probably be considered to identify new sources of resistance to plant viruses. Part of the methodology described here could alternatively be used in TILLING experiments that serve to generate new eIF4E alleles.     

两系杂交小麦恢复系MR168抗条锈病基因遗传分析及分子标记定位

小麦条锈病是影响杂交小麦普及推广的重要因素。文章利用基因推导法和SSR分子标记技术,研究了温光型两系杂交小麦恢复系MR168的抗条锈性遗传规律及其控制基因染色体位置。结果表明,MR168对CY29、CY31、CY32、CY33等条锈菌生理小种表现高抗至免疫;对SY95-71/MR168杂交组合的正反交F1、BC1、F2和F3群体分单株接种鉴定显示,MR168对CY32号小种的抗性受1对显性核基因控制,该抗病基因来源于春小麦品种辽春10号。利用集群分离分析法(Bulked segregant analysis,BSA)和简单重复序列(Simple sequence repeat,SSR)分子标记分析抗病亲本MR168、感病亲本SY95-71及183个F2代单株,发现了与MR168抗条锈病基因连锁的5个微卫星标记Xgwm273、Xgwm18、Xbarc187、Xwmc269、Xwmc406,并将该基因初步定位在1BS着丝粒附近,暂命名为YrMR168;构建了包含YrMR168的SSR标记遗传图谱,距离YrMR168最近的两个微卫星位点是Xgwm18和Xbarc187,遗传距离分别为1.9 cM和2.4 cM,这两个微卫星标记可用于杂交小麦抗条锈病分子标记辅助育种。     

Molecular mapping of stripe rust resistance gene YrSN104 in Chinese wheat line Shaannong 104

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a serious yield-limiting factor for wheat production worldwide. The objective of this study was to identify and map a stripe rust resistance gene in wheat line Shaannong 104 using SSR markers. F(1) , F(2) and F(3) populations from Shaannong 104/Mingxian 169 were inoculated with Chinese Pst race CYR32 in a greenhouse. Shaannong 104 carried a single dominant gene, YrSN104. Six potential polymorphic SSR markers identified in bulk segregant analysis were used to genotype F(2) and F(3) families. YrSN104 was closely linked with all six SSR markers on chromosome 1BS with genetic distances of 2.0 cM (Xgwm18, Xgwm273, Xbarc187), 2.6 cM (Xgwm11, Xbarc137) and 5.9 cM (Xbarc240). Pedigree analysis, pathogenicity tests using 26 Pst races, haplotyping of associated markers on isogenic lines carrying known stripe rust resistance genes, and associations with markers suggested that YrSN104 was a new resistance gene or an allele at the Yr24/Yr26 locus on chromosome 1BS. Deployment of YrSN104 singly or in combination to elite genotypes could play an effective role to lessen yield losses caused by stripe rust.     

TaADF4, an actin‐depolymerizing factor from wheat,is required for resistance to the stripe rust pathogen Puccinia striiformis f. sp. tritici

Actin filament assembly in plants is a dynamic process, requiring the activity of more than 75 actin‐binding proteins. Central to the regulation of filament assembly and stability is the activity of a conserved family of actin‐depolymerizing factors (ADFs), whose primarily function is to regulate the severing and depolymerization of actin filaments. In recent years, the activity of ADF proteins has been linked to a variety of cellular processes, including those associated with response to stress. Herein, a wheat ADF gene, TaADF4, was identified and characterized. TaADF4 encodes a 139‐amino‐acid protein containing five F‐actin‐binding sites and two G‐actin‐binding sites, and interacts with wheat (Triticum aestivum) Actin1 (TaACT1), in planta. Following treatment of wheat, separately, with jasmonic acid, abscisic acid or with the avirulent race, CYR23, of the stripe rust pathogen Puccinia striiformis f. sp. tritici, we observed a rapid induction in accumulation of TaADF4 mRNA. Interestingly, accumulation of TaADF4 mRNA was diminished in response to inoculation with a virulent race, CYR31. Silencing of TaADF4 resulted in enhanced susceptibility to CYR23, demonstrating a role for TaADF4 in defense signaling. Using a pharmacological‐based approach, coupled with an analysis of host response to pathogen infection, we observed that treatment of plants with the actin‐modifying agent latrunculin B enhanced resistance to CYR23, including increased production of reactive oxygen species and enhancement of localized hypersensitive cell death. Taken together, these data support the hypothesis that TaADF4 positively modulates plant immunity in wheat via the modulation of actin cytoskeletal organization.     

Comparative sequence and structure analysis of eIF1A and eIF1AD

Background

Eukaryotic translation initiation factor 1A (eIF1A) is universally conserved in all organisms. It has multiple functions in translation initiation, including assembly of the ribosomal pre-initiation complexes, mRNA binding, scanning, and ribosomal subunit joining. eIF1A binds directly to the small ribosomal subunit, as well as to several other translation initiation factors. The structure of an eIF1A homolog, the eIF1A domain-containing protein (eIF1AD) was recently determined but its biological functions are unknown. Since eIF1AD has a known structure, as well as a homolog, whose structure and functions have been extensively studied, it is a very attractive target for sequence and structure analysis.

Results

Structure/sequence analysis of eIF1AD found significant conservation in the surfaces corresponding to the ribosome-binding surfaces of its paralog eIF1A, including a nearly invariant surface-exposed tryptophan residue, which plays an important role in the interaction of eIF1A with the ribosome. These results indicate that eIF1AD may bind to the ribosome, similar to its paralog eIF1A, and could have roles in ribosome biogenenesis or regulation of translation. We identified conserved surfaces and sequence motifs in the folded domain as well as the C-terminal tail of eIF1AD, which are likely protein-protein interaction sites. The roles of these regions for eIF1AD function remain to be determined. We have also identified a set of trypanosomatid-specific surface determinants in eIF1A that could be a promising target for development of treatments against these parasites.

Conclusions

The results described here identify regions in eIF1A and eIF1AD that are likely to play major functional roles and are promising therapeutic targets. Our findings and hypotheses will promote new research and help elucidate the functions of eIF1AD.

     

Molecular mapping of stripe rust resistance gene <Emphasis Type="Italic">YrHu</Emphasis> derived from <Emphasis Type="Italic">Psathyrostachys huashanica</Emphasis>

Wheat stripe rust is a destructive disease that affects most wheat-growing areas worldwide. Resistance genes from related species and genera add to the genetic diversity available to wheat breeding programs. The stripe rust-resistant introgression line H9020-17-25-6-4 was developed from a cross of resistant Psathyrostachys huashanica with the susceptible wheat cultivar 7182. H9020-17-25-6-4 is resistant to all existing Chinese stripe rust races, including the three most widely virulent races, CYR32, CYR33, and V26. We attempted to characterize this new line by genomic in situ hybridization (GISH) and genetic analysis. GISH using P. huashanica genomic DNA as a probe indicated that the translocated segment was too small to be detected. Genetic analysis involving F₁, F₂, and F_2:3 materials derived from a cross of Mingxian 169 and H9020-17-25-6-4 indicated that a single dominant gene from H9020-17-25-6-4, temporarily designated YrHu, conferred resistance to CYR29 and CYR33. A genetic map consisting of four simple sequence repeat, two sequence-tagged site (STS), and two sequence-related amplified polymorphism markers was constructed. YrHu was located on the short arm of chromosome 3A and was about 0.7 and 1.5 cM proximal to EST-STS markers BG604577 and BE489244, respectively. Both the gene and the closely linked markers could be used in marker-assisted selection.     

Genetic analyses of BaMMV/BaYMV resistance in barley accession HOR4224 result in the identification of an allele of the translation initiation factor 4e (Hv-eIF4E) exclusively effective against Barley mild mosaic virus (BaMMV)

Key message

Based on a strategy combining extensive segregation analyses and tests for allelism with allele-specific re-sequencing an Hv-eIF4E allele exclusively effective against BaMMV was identified and closely linked markers for BaYMV resistance were developed.

Abstract

Soil-borne barley yellow mosaic disease is one of the most important diseases of winter barley. In extensive screenings for resistance, accession ‘HOR4224’ being resistant to three strains of Barley mild mosaic virus (BaMMV-ASL1, BaMMV-Sil, and BaMMV-Teik) and two strains of Barley yellow mosaic virus (BaYMV-1 and BaYMV-2) was identified. Analyses using Bmac29, being to some extent diagnostic for the rym4/5 locus, gave hint to the presence of the susceptibility-encoding allele at this locus. Therefore, 107 DH lines derived from the cross ‘HOR4224’ × ‘HOR10714’ (susceptible) were screened for resistance. Genetic analyses revealed an independent inheritance of resistance to BaMMV and BaYMV ( $\chi_{1:1:1:1}^{2}$  = 5.58) both encoded by a single gene (BaMMV $\chi_{1:1}^{2}$  = 0.477; BaYMV $\chi_{1:1}^{2}$  = 0.770). Although Bmac29 indicated the susceptibility-encoding allele, BaMMV resistance of ‘HOR4224’ co-localized with rym4/rym5. The BaYMV resistance was mapped to chromosome 5H in the region of rym3. Sequencing of full length cDNA of the Hv-eIF4E gene displayed an already sequenced allele described to be efficient against BaMMV and BaYMV. However, the F₁ progenies of crosses involving ‘HOR4224’ and rym4/rym5 donors were all resistant to BaMMV but susceptible to BaYMV. Therefore, this is the first report of an allele at the rym4/rym5 locus exclusively efficient against BaMMV. Changes in the specificity are due to one non-synonymous amino acid substitution (I118K). Results obtained elucidate that combining extensive segregation analyses and tests for allelism involving different strains of BaMMV/BaYMV in combination with allele-specific re-sequencing is an efficient strategy for gene and allele detection in complex pathosystems.     

Mapping resistance to the bird cherry-oat aphid and the greenbug in wheat using sequence-based genotyping

Key message

Identification of novel resistance QTL against wheat aphids. First QTL-resistance report for R. padi in wheat and chromosome 2DL for S. graminum . These sources have potential use in wheat breeding.

Abstract

The aphids Rhopalosiphum padi and Schizaphis graminum are important pests of common wheat (Triticum aestivum L.). Characterization of the genetic bases of resistance sources is crucial to facilitate the development of resistant wheat cultivars to these insects. We examined 140 recombinant inbred lines (RILs) from the cross of Seri M82 wheat (susceptible) with the synthetic hexaploid wheat CWI76364 (resistant). RILs were phenotyped for R. padi antibiosis and tolerance traits. Phenotyping of S. graminum resistance was based on leaf chlorosis in a greenhouse screening and the number of S. graminum/tiller in the field. RILs were also scored for pubescence. Using a sequence-based genotyping method, we located genomic regions associated with these resistance traits. A quantitative trait locus (QTL) for R. padi antibiosis (QRp.slu.4BL) that explained 10.2 % of phenotypic variation was found in chromosome 4BL and located 14.6 cM apart from the pubescence locus. We found no association between plant pubescence and the resistance traits. We found two QTLs for R. padi tolerance (QRp.slu.5AL and QRp.slu.5BL) in chromosomes 5AL and 5BL, with an epistatic interaction between a locus in chromosome 3AL (EnQRp.slu.5AL) and QRp.slu.5AL. These genomic regions explained about 35 % of the phenotypic variation. We re-mapped a previously reported gene for S. graminum resistance (putatively Gba) in 7DL and found a novel QTL associated with the number of aphids/tiller (QGb.slu-2DL) in chromosome 2DL. This is the first report on the genetic mapping of R. padi resistance in wheat and the first report where chromosome 2DL is shown to be associated with S. graminum resistance.     

Crystal structure of the eukaryotic translation initiation factor 2A from Schizosaccharomyces pombe

The eukaryotic translation initiation factor 2A (eIF2A) was identified as a factor that stimulates the binding of methionylated initiator tRNA (Met-tRNA _i ^Met ) to the 40S ribosomal subunit, but its physiological role remains poorly defined. Recently, eIF2A was shown to be involved in unconventional translation initiation from CUG codons and in viral protein synthesis under stress conditions where eIF2 is inactivated. We determined the crystal structure of the WD-repeat domain of Schizosaccharomyces pombe eIF2A at 2.5 Å resolution. The structure adopts a novel nine-bladed β-propeller fold. In contrast to the usual β-propeller proteins, the central channel of the molecule has the narrower opening on the bottom of the protein and the wider opening on the top. Highly conserved residues are concentrated in the positively-charged top face, suggesting the importance of this face for interactions with nucleic acids or other initiation factors.