Optimizing an integrated biovigilance toolbox to study the spatial distribution and dynamic changes of airborne mycobiota, with a focus on cereal rust fungi in western Canada.

In the face of evolving agricultural practices and climate change, tools towards an integrated biovigilance platform to combat crop diseases, spore sampling, DNA diagnostics and predictive trajectory modelling were optimized. These tools revealed microbial dynamics and were validated by monitoring cereal rust fungal pathogens affecting wheat, oats, barley and rye across four growing seasons (2015-2018) in British Columbia and during the 2018 season in southern Alberta. ITS2 metabarcoding revealed disparity in aeromycobiota diversity and compositional structure across the Canadian Rocky Mountains, suggesting a barrier effect on air flow and pathogen dispersal. A novel bioinformatics classifier and curated cereal rust fungal ITS2 database, corroborated by real-time PCR, enhanced the precision of cereal rust fungal species identification. Random Forest modelling identified crop and land-use diversification as well as atmospheric pressure and moisture as key factors in rust distribution. As a valuable addition to explain observed differences and patterns in rust fungus distribution, trajectory HYSPLIT modelling tracked rust fungal urediniospores' northeastward dispersal from the Pacific Northwest towards southern British Columbia and Alberta, indicating multiple potential origins. Our Canadian case study exemplifies the power of an advanced biovigilance toolbox towards developing an early-warning system for farmers to detect and mitigate impending disease outbreaks.

Distinguishing Puccinia striiformis f. sp. tritici Isolates Using Genomic Sequencing: A Case Study.

We are reporting on the utilization of high-throughput sequencing and different sequencing analysis tools to delineate identification of different isolates of the stripe rust fungal pathogen Puccinia striiformis f. sp. tritici (Pst). Different approaches are shown: utilization of rDNA sequences and random sequences that may be very useful to make sure that isolates belong to Pst and to distinguished closely related isolates. Identification of unique/lost sequences could lead to the identification of effectors associated with specific isolates.

Association mapping of autumn-seeded rye (Secale cereale L.) reveals genetic linkages between genes controlling winter hardiness and plant development.

Winter field survival (WFS) in autumn-seeded winter cereals is a complex trait associated with low temperature tolerance (LTT), prostrate growth habit (PGH), and final leaf number (FLN). WFS and the three sub-traits were analyzed by a genome-wide association study of 96 rye (Secale cereal L.) genotypes of different origins and winter-hardiness levels. A total of 10,244 single nucleotide polymorphism (SNP) markers were identified by genotyping by sequencing and 259 marker-trait-associations (MTAs; p < 0.01) were revealed by association mapping. The ten most significant SNPs (p < 1.49e-04) associated with WFS corresponded to nine strong candidate genes: Inducer of CBF Expression 1 (ICE1), Cold-regulated 413-Plasma Membrane Protein 1 (COR413-PM1), Ice Recrystallization Inhibition Protein 1 (IRIP1), Jasmonate-resistant 1 (JAR1), BIPP2C1-like protein phosphatase, Chloroplast Unusual Positioning Protein-1 (CHUP1), FRIGIDA-like 4 (FRL4-like) protein, Chalcone Synthase 2 (CHS2), and Phenylalanine Ammonia-lyase 8 (PAL8). Seven of the candidate genes were also significant for one or several of the sub-traits supporting the hypothesis that WFS, LTT, FLN, and PGH are genetically interlinked. The winter-hardy rye genotypes generally carried additional allele variants for the strong candidate genes, which suggested allele diversity was a major contributor to cold acclimation efficiency and consistent high WFS under varying field conditions.

The Relationships between Plant Developmental Traits and Winter Field Survival in Rye (Secale cereale L.).

Overwintering cereals accumulate low temperature tolerance (LTT) during cold acclimation in the autumn. Simultaneously, the plants adjust to the colder season by making developmental changes at the shoot apical meristem. These processes lead to higher winter hardiness in winter rye varieties (Secale cereale L.) adapted to Northern latitudes as compared to other cereal crops. To dissect the winter-hardiness trait in rye, a panel of 96 genotypes of different origins and growth habits was assessed for winter field survival (WFS), LTT, and six developmental traits. Best Linear Unbiased Estimates for WFS determined from five field trials correlated strongly with LTT (r = 0.90, p < 0.001); thus, cold acclimation efficiency was the major contributor to WFS. WFS also correlated strongly (p < 0.001) with final leaf number (r = 0.80), prostrate growth habit (r = 0.61), plant height (r = 0.34), but showed weaker associations with top internode length (r = 0.30, p < 0.01) and days to anthesis (r = 0.25, p < 0.05). The heritability estimates (h2) for WFS-associated traits ranged from 0.45 (prostrate growth habit) to 0.81 (final leaf number) and were overall higher than for WFS (h2 = 0.48). All developmental traits associated with WFS and LTT are postulated to be regulated by phytohormone levels at shoot apical meristem.

Identification and expression analysis of some wheat F-box subfamilies during plant development and infection by Puccinia triticina.

As one of the largest protein families in plants, F-box proteins are involved in many important cellular processes. Until now, a limited number of investigations have been conducted on wheat F-box genes due to its variable structure and large and polyploid genome. Classification, identification, structural analysis, evolutionary relationship, and chromosomal distribution of some wheat F-box genes are described in the present study. A total number of 1013 potential F-box proteins which are encoded by 409 genes was identified in wheat, and classified into 12 subfamilies based on their C-terminal domain structures. Furthermore, proteins with identical or similar C-terminal domain were clustered together. Location of 409 F-box genes was identified on all 21 wheat chromosomes but showed an uneven distribution. Segmental duplication was the main reason for the increase in the number of wheat F-box genes. Gene expression analysis based on digital PCR showed that most of the F-box genes were highly expressed in the later development stages of wheat, including the formation of spike, grain, flag leaf, and participated in drought stress (DS), heat stress (HS), and their combination (HD). Of the nine F-box genes we investigated using quantitative PCR (qPCR) following fungal pathogen infection, five were involved in wheat resistance to the infection by leaf rust pathogen and one in the susceptible response. These results provide important information on wheat F-box proteins for further functional studies, especially the proteins that played roles in response to heat and drought stresses and leaf rust pathogen infection.

A triticale tapetal non-specific lipid transfer protein (nsLTP) is translocated to the pollen cell wall.

KEY MESSAGE: A Triticeae type III non-specific lipid transfer protein (nsLTP) was shown for the first time to be translocated from the anther tapetum to the pollen cell wall. Two anther-expressed non-specific lipid transfer proteins (nsLTPs) were identified in triticale (× Triticosecale Wittmack). LTPc3a and LTPc3b contain a putative signal peptide sequence and eight cysteine residues in a C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C pattern. These proteins belong to the type III class of nsLTPs which are expressed exclusively in the inflorescence of angiosperms. The level of LTPc3 transcript in the anther was highest at the tetrad and uninucleate microspore stages, and absent in mature pollen. In situ hybridization showed that LTPc3 was expressed in the tapetal layer of the developing triticale anther. The expression of the LTPc3 protein peaked at the uninucleate microspore stage, but was also found to be associated with the mature pollen. Accordingly, an LTPc3a::GFP translational fusion expressed in transgenic Brachypodium distachyon first showed activity in the tapetum, then in the anther locule, and later on the mature pollen grain. Altogether, these results represent the first detailed characterization of a Triticeae anther-expressed type III nsLTP with possible roles in pollen cell wall formation.

Analysis of MAPK and MAPKK gene families in wheat and related Triticeae species.

BACKGROUND: The mitogen-activated protein kinase (MAPK) family is involved in signal transduction networks that underpin many different biological processes in plants, ranging from development to biotic and abiotic stress responses. To date this class of enzymes has received little attention in Triticeae species, which include important cereal crops (wheat, barley, rye and triticale) that represent over 20% of the total protein food-source worldwide. RESULTS: The work presented here focuses on two subfamilies of Triticeae MAPKs, the MAP kinases (MPKs), and the MAPK kinases (MKKs) whose members phosphorylate the MPKs. In silico analysis of multiple Triticeae sequence databases led to the identification of 152 MAPKs belonging to these two sub-families. Some previously identified MAPKs were renamed to reflect the literature consensus on MAPK nomenclature. Two novel MPKs, MPK24 and MPK25, have been identified, including the first example of a plant MPK carrying the TGY activation loop sequence common to mammalian p38 MPKs. An EF-hand calcium-binding domain was found in members of the Triticeae MPK17 clade, a feature that appears to be specific to Triticeae species. New insights into the novel MEY activation loop identified in MPK11s are offered. When the exon-intron patterns for some MPKs and MKKs of wheat, barley and ancestors of wheat were assembled based on transcript data in GenBank, they showed deviations from the same sequence predicted in Ensembl. The functional relevance of MAPKs as derived from patterns of gene expression, MPK activation and MKK-MPK interaction is discussed. CONCLUSIONS: A comprehensive resource of accurately annotated and curated Triticeae MPK and MKK sequences has been created for wheat, barley, rye, triticale, and two ancestral wheat species, goat grass and red wild einkorn. The work we present here offers a central information resource that will resolve existing confusion in the literature and sustain expansion of MAPK research in the crucial Triticeae grains.

High Density Single Nucleotide Polymorphism (SNP) Mapping and Quantitative Trait Loci (QTL) Analysis in a Biparental Spring Triticale Population Localized Major and Minor Effect Fusarium Head Blight Resistance and Associated Traits QTL.

Triticale (xTriticosecale Wittmack) is an important feed crop which suffers severe yield, grade and end-use quality losses due to Fusarium head blight (FHB). Development of resistant triticale cultivars is hindered by lack of effective genetic resistance sources. To dissect FHB resistance, a doubled haploid spring triticale population produced from the cross TMP16315/AC Ultima using a microspore culture method, was phenotyped for FHB incidence, severity, visual rating index (VRI), deoxynivalenol (DON) and some associated traits (ergot, grain protein content, test weight, yield, plant height and lodging) followed by single nucleotide polymorphism (SNP) genotyping. A high-density map consisting of 5274 SNPs, mapped on all 21 chromosomes with a map density of 0.48 cM/SNP, was constructed. Together, 17 major quantitative trait loci were identified for FHB on chromosomes 1A, 2B, 3A, 4A, 4R, 5A, 5R and 6B; two of incidence loci (on 2B and 5R) also co-located with loci for severity and VRI, and two other loci of VRI (on 1A and 4R) with DON accumulation. Major and minor loci were also identified for all other traits in addition to many epistasis loci. This study provides new insight into the genetic basis of FHB resistance and their association with other traits in triticale.

Investigating Triticeae anther gene promoter activity in transgenic Brachypodium distachyon.

MAIN CONCLUSION: In this report, we demonstrate that Brachypodium distachyon could serve as a relatively high throughput in planta functional assay system for Triticeae anther-specific gene promoters. There remains a vast gap in our knowledge of the promoter cis-acting elements responsible for the transcriptional regulation of Triticeae anther-specific genes. In an attempt to identify conserved cis-elements, 14 pollen-specific and 8 tapetum-specific Triticeae putative promoter sequences were analyzed using different promoter sequence analysis tools. Several cis-elements were found to be enriched in these sequences and their possible role in gene expression regulation in the anther is discussed. Despite the fact that potential cis-acting elements can be identified within putative promoter sequence datasets, determining whether particular promoter sequences can in fact direct proper tissue-specific and developmental gene expression still needs to be confirmed via functional assays preferably performed in closely related plants. Transgenic functional assays with Triticeae species remain challenging and Brachypodium distachyon may represent a suitable alternative. The promoters of the triticale pollen-specific genes group 3 pollen allergen (PAL3) and group 4 pollen allergen (PAL4), as well as the tapetum-specific genes chalcone synthase-like 1 (CHSL1), from wheat and cysteine-rich protein 1 (CRP1) from triticale were fused to the green fluorescent protein gene (GFP) and analyzed in transgenic Brachypodium. This report demonstrates that this model species could serve to accelerate the functional analysis of Triticeae anther-specific gene promoters.

Identification and characterization of rye genes not expressed in allohexaploid triticale.

BACKGROUND: One of the most important evolutionary processes in plants is polyploidization. The combination of two or more genomes in one organism often initially leads to changes in gene expression and extensive genomic reorganization, compared to the parental species. Hexaploid triticale (x Triticosecale) is a synthetic hybrid crop species generated by crosses between T. turgidum and Secale cereale. Because triticale is a recent synthetic polyploid it is an important model for studying genome evolution following polyploidization. Molecular studies have demonstrated that genomic sequence changes, consisting of sequence elimination or loss of expression of genes from the rye genome, are common in triticale. High-throughput DNA sequencing allows a large number of genes to be surveyed, and transcripts from the different homeologous copies of the genes that have high sequence similarity can be better distinguished than hybridization methods previously employed. RESULTS: The expression levels of 23,503 rye cDNA reference contigs were analyzed in 454-cDNA libraries obtained from anther, root and stem from both triticale and rye, as well as in five 454-cDNA data sets created from triticale seedling shoot, ovary, stigma, pollen and seed tissues to identify the classes of rye genes silenced or absent in the recent synthetic hexaploid triticale. Comparisons between diploid rye and hexaploid triticale detected 112 rye cDNA contigs (~0.5%) that were totally undetected by expression analysis in all triticale tissues, although their expression was relatively high in rye tissues. Non-expressed rye genes were found to be strikingly less similar to their closest BLASTN matches in the wheat genome or in the other Triticum genomes than a test set of 200 random rye genes. Genes that were not detected in the RNA-seq data were further characterized by testing for their presence in the triticale genome by PCR using genomic DNA as a template. CONCLUSION: Genes with low similarity between rye sequences and their closest matches in the Triticum genome have a higher probability to be repressed or absent in the allopolyploid genome.

Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding.

Marker-assisted selection (MAS) refers to the use of molecular markers to assist phenotypic selections in crop improvement. Several types of molecular markers, such as single nucleotide polymorphism (SNP), have been identified and effectively used in plant breeding. The application of next-generation sequencing (NGS) technologies has led to remarkable advances in whole genome sequencing, which provides ultra-throughput sequences to revolutionize plant genotyping and breeding. To further broaden NGS usages to large crop genomes such as maize and wheat, genotyping-by-sequencing (GBS) has been developed and applied in sequencing multiplexed samples that combine molecular marker discovery and genotyping. GBS is a novel application of NGS protocols for discovering and genotyping SNPs in crop genomes and populations. The GBS approach includes the digestion of genomic DNA with restriction enzymes followed by the ligation of barcode adapter, PCR amplification and sequencing of the amplified DNA pool on a single lane of flow cells. Bioinformatic pipelines are needed to analyze and interpret GBS datasets. As an ultimate MAS tool and a cost-effective technique, GBS has been successfully used in implementing genome-wide association study (GWAS), genomic diversity study, genetic linkage analysis, molecular marker discovery and genomic selection under a large scale of plant breeding programs.

The stripe rust resistance gene Yr10 encodes an evolutionary-conserved and unique CC-NBS-LRR sequence in wheat.

The first seedling or all-stage resistance (R) R gene against stripe rust isolated from Moro wheat (Triticum aestivum L.) using a map-based cloning approach was identified as Yr10. Clone 4B of this gene encodes a highly evolutionary-conserved and unique CC-NBS-LRR sequence. Clone 4E, a homolog of Yr10, but lacking transcription start site (TSS) and putative TATA-box and CAAT-box, is likely a non-expressed pseudogene. Clones 4B and 4E are 84% identical and divergent in the intron and the LRR domain. Gene silencing and transgenesis were used in conjunction with inoculation with differentially avirulent and virulent stripe rust strains to demonstrate Yr10 functionality. The Yr10 CC-NBS-LRR sequence is unique among known CC-NBS-LRR R genes in wheat but highly conserved homologs (E = 0.0) were identified in Aegilops tauschii and other monocots including Hordeum vulgare and Brachypodium distachyon. Related sequences were also identified in genomic databases of maize, rice, and in sorghum. This is the first report of a CC-NBS-LRR resistance gene in plants with limited homologies in its native host, but with numerous homologous R genes in related monocots that are either host or non-hosts for stripe rust. These results represent a unique example of gene evolution and dispersion across species.

Characterization of the caleosin gene family in the Triticeae.

BACKGROUND: The caleosin genes encode proteins with a single conserved EF hand calcium-binding domain and comprise small gene families found in a wide range of plant species. Some members of the gene family have been shown to be upregulated by environmental stresses including low water availability and high salinity. Caleosin 3 from wheat has been shown to interact with the α-subunit of the heterotrimeric G proteins, and to act as a GTPase activating protein (GAP). This study characterizes the size and diversity of the gene family in wheat and related species and characterizes the differential tissue-specific expression of members of the gene family. RESULTS: A total of 34 gene family members that belong to eleven paralogous groups of caleosins were identified in the hexaploid bread wheat, T. aestivum. Each group was represented by three homeologous copies of the gene located on corresponding homeologous chromosomes, except the caleosin 10, which has four gene copies. Ten gene family members were identified in diploid barley, Hordeum vulgare, and in rye, Secale cereale, seven in Brachypodium distachyon, and six in rice, Oryza sativa. The analysis of gene expression was assayed in triticale and rye by RNA-Seq analysis of 454 sequence sets and members of the gene family were found to have diverse patterns of gene expression in the different tissues that were sampled in rye and in triticale, the hybrid hexaploid species derived from wheat and rye. Expression of the gene family in wheat and barley was also previously determined by microarray analysis, and changes in expression during development and in response to environmental stresses are presented. CONCLUSIONS: The caleosin gene family had a greater degree of expansion in the Triticeae than in the other monocot species, Brachypodium and rice. The prior implication of one member of the gene family in the stress response and heterotrimeric G protein signaling, points to the potential importance of the caleosin gene family. The complexity of the family and differential expression in various tissues and under conditions of abiotic stress suggests the possibility that caleosin family members may play diverse roles in signaling and development that warrants further investigation.

Developmental transcriptional profiling reveals key insights into Triticeae reproductive development.

Despite their importance, there remains a paucity of large-scale gene expression-based studies of reproductive development in species belonging to the Triticeae. As a first step to address this deficiency, a gene expression atlas of triticale reproductive development was generated using the 55K Affymetrix GeneChip(®) wheat genome array. The global transcriptional profiles of the anther/pollen, ovary and stigma were analyzed at concurrent developmental stages, and co-expressed as well as preferentially expressed genes were identified. Data analysis revealed both novel and conserved regulatory factors underlying Triticeae floral development and function. This comprehensive resource rests upon detailed gene annotations, and the expression profiles are readily accessible via a web browser.

Carbohydrate profiling in seeds and seedlings of transgenic triticale modified in the expression of sucrose:sucrose-1-fructosyltransferase (1-SST) and sucrose:fructan-6-fructosyltransferase (6-SFT).

Constructs with sucrose-sucrose 1-fructosyltransferase (1-SST) from rye and or sucrose-fructan 6-fructosyltransferase (6-SFT) from wheat were placed under the control of wheat aleurone-specific promoter and expressed in triticale using biolistic and microspore transformation. Transgenic lines expressing one or both the 1-SST and the 6-SFT accumulated 50% less starch and 10-20 times more fructan, particularly 6-kestose, in the dry seed compared to the untransformed wild-type (WT) triticale; other fructans ranged in size from DP 4 to DP 15. During germination from 1 to 4 days after imbibition (dai), fructans were rapidly metabolized and only in transgenic lines expressing both 1-SST and 6-SFT were fructan contents significantly higher than in the untransformed controls after 4 days. In situ hybridization confirmed expression of 6-SFT in the aleurone layer in imbibed seeds of transformed plants. When transgenic lines were subjected to a cold stress of 4°C for 2 days, synthesis of fructan increased compared to untransformed controls during low-temperature germination. The increase of fructan in dry seed and germinating seedling was generally associated with transcript expression levels in transformed plants but total gene expression was not necessarily correlated with the time course accumulation of fructan during germination. This is the first report of transgenic modification of cereals to achieve production of fructans in cereal seeds and during seed germination.

A molecular and proteomic investigation of proteins rapidly released from triticale pollen upon hydration.

Analysis of Triticale (×Triticosecale Wittmack cv. AC Alta) mature pollen proteins quickly released upon hydration was performed using two-dimensional gel electrophoresis followed by mass spectrometry. A total of 17 distinct protein families were identified and these included expansins, profilins, and various enzymes, many of which are pollen allergens. The corresponding genes were obtained and expression studies revealed that the majority of these genes were only expressed in developing anthers and pollen. Some genes including glucanase, glutathione peroxidase, glutaredoxin, and a profilin were found to be widely expressed in different reproductive and vegetative tissues. Group 11 pollen allergens, polygalacturonase, and actin depolymerizing factor were characterized for the first time in the Triticeae. This study represents a distinctive combination of proteomic and molecular analyses of the major cereal pollen proteins released upon hydration and therefore at the forefront of pollen-stigma interactions.

Management of women with surgically staged 1 uterine papillary serous cancer.

Objective. To review the management and outcomes of women with surgically staged 1 UPSC. Methods. We report on a case series from 2008-2010 from Hamilton Canada. We summarize the data from a literature search on surgically staged 1 UPSC. Results. There is a group women with Stage 1A UPSC with no residual disease at time of surgery who do not require adjuvant therapy. Vault recurrences appear to be lower in women who received adjuvant vault radiation. Chemotherapy appears to confer longer survival for those women with Stage 1B or 1C disease compared of those observed or who had radiation alone. Conclusion. Adjuvant therapy appears to confer benefit in certain groups of women with stage 1 UPSC. A randomized controlled study would clarify the degree of benefit.

Revealing plant defense signaling: getting more sophisticated with phosphoproteomics.

The regulation mechanisms of any plant-pathogen interaction are complex and dynamic. A proteomic approach is necessary in understanding regulatory networks because it identifies new proteins in relation to their function and ultimately aims to clarify how their expression, accumulation and modification is controlled. One of the major control mechanisms for protein activity in plant-pathogen interactions is protein phosphorylation, and an understanding of the significance of protein phosphorylation in plant-pathogen interaction can be overwhelming. Due to the high number of protein kinases and phosphatases in any single plant genome and specific limitations of any technologies, it is extremely challenging for us to fully delineate the phosphorylation machinery. Current proteomic approaches and technology advances have demonstrated their great potential in identifying new components. Recent studies in well-developed plant-pathogen systems have revealed novel phosphorylation pathways, and some of them are off the core phosphorylation cascades. Additional phosphoproteomic studies are needed to increase our comprehension of the different mechanisms and their fine tuning involved in the host resistance response to pathogen attacks.

Development and assessment of DArT markers in triticale.

Triticale (X Triticosecale Wittm.) is a hybrid derived by crossing wheat (Triticum sp.) and rye (Secale sp.). Till date, only a limited number of simple sequence repeat (SSRs) markers have been used in triticale molecular analyses and there is a need to identify dedicated high-throughput molecular markers to better exploit this crop. The objective of this study was to develop and evaluate diversity arrays technology (DArT) markers in triticale. DArT marker technology offers a high level of multiplexing. Development of new markers from triticale accessions was combined with mining the large collection of previously developed markers in rye and wheat. Three genotyping arrays were used to analyze a collection of 144 triticale accessions. The polymorphism level ranged from 8.6 to 23.8% for wheat and rye DArT markers, respectively. Among the polymorphic markers, rye markers were the most abundant (3,109) followed by wheat (2,214) and triticale (719). The mean polymorphism information content values were 0.34 for rye DArT markers and 0.37 for those from triticale and wheat. High correlation was observed between similarity matrices derived from rye, triticale, wheat and combined marker sets, as well as for the cophenetic values matrices. Cluster analysis revealed genetic relationships among the accessions consistent with the agronomic and pedigree information available. The newly developed triticale DArT markers as well as those originated from rye and wheat provide high quality markers that can be used for diversity analyses and might be exploited in a range of molecular breeding and genomics applications in triticale.

Overexpression of PAX5 induces apoptosis in multiple myeloma cells.

PAX5 is an essential transcription factor for the commitment of lymphoid progenitors to the B-lymphocyte lineage. PAX5 suppression results in retrodifferentiation of B lymphocytes to an uncommitted progenitor cell stage, whereas PAX5 suppression in mature B lymphocytes leads to further development into plasma cells. Here, we have analyzed the fate of plasma cell lines following PAX5 reexpression. Human B cell lines were infected with Ad5/F35 adenoviruses encoding either EYFP or PAX5. Expression analysis of specific plasma cell transcription factors (IRF4, Blimp-1 and XBP-1) suggests that PAX5 reexpression does not induce retrodifferentiation of plasma cells into B lymphocytes. Interestingly, the viability of RPMI-8226 and U266 multiple myeloma cell lines markedly declined at 4-7 days post-transduction, whereas other plasma cell lines maintained their viability. Apoptosis analysis through Annexin V measurement also revealed a higher level of apoptosis in PAX5-expressing myeloma cell lines. Finally, Western blot analysis of pro- and anti-apoptotic proteins revealed that the anti-apoptotic protein MCL-1 was down-modulated in PAX5-transduced multiple myeloma cell lines. In conclusion, our results show that the expression of PAX5 in plasma cell lines induces apoptosis exclusively in multiple myelomas. This might represent a potential therapeutic avenue in the treatment of multiple myeloma.