African rice ( Oryza glaberrima ) was independently domesticated in West Africa around 3000 years ago, and has long been intertwined in the history of the region. The gradual replacement of African rice by Asian rice ( Oryza sativa ), which was introduced when European settlers arrived, has since dominated rice cultivation in Africa. Domesticated rice species are affected by bacterial leaf blight (BLB), which is caused by the pathogen Xanthomonas oryzae pv. oryzae (Xoo). Here we provide evidence that the bacterial leaf blight pathogen in Africa (AfXoo) belongs to a distinct phylogroup from the one circulating in Asia (AsXoo), and has a different evolutionary history. Analysis of 88 AfXoo genomes identified five groups, one of which is a highly diverse population that might have probably given rise to three independent clonal populations based on multiple genetic tests. Tip-dating analysis revealed that the emergence and expansion of AfXoo coincided with the rise and fall of African rice nearly a thousand years ago, and O. sativa served as a bottleneck in the evolution of AfXoo over time. Although the type III effectors (T3E), proteins that are secreted by the pathogen to evade host resistance or seize control of host nutrients, are highly conserved in AfXoo, we observed some variation in effector families. Different evolutionary modifications in the transcription activator-like effectors (TALEs), especially in repeat variable di-residues (RVDs), likely enabled adaptation to both host species. Previous analyses carried out on samples collected in Burkina Faso have shown that there could be more than one TALE repertoire combination in the field, and genome sequencing data revealed potential TALE evolutionary mechanisms that could happen. Our research provides a comprehensive genetic history of bacterial blight in West Africa, and its past and present impact on rice cultivation in the region. For thousands of years, rice cultivation has been an integral part of African agriculture. However, the cultivation of the locally domesticated African rice cultivar ( Oryza glaberrima ) has been gradually shifted towards Asian rice varieties ( Oryza sativa ), which has affected the adaptation of the native pathogen population. One of these pathogens is the causal agent of bacterial leaf blight, Xanthomonas oryzae pv. oryzae ( Xoo ). Here we performed a population genomics approach to understand the evolutionary history and virulence spectrum of African Xoo (AfXoo), a unique phylogroup within the Xanthomonas oryzae species. Our results suggest that AfXoo were first adapted to African rice at least a thousand years ago. The introduction of O. sativa has shaped the recent population dynamics of AfXoo. TALEs are tightly conserved in AfXoo with multiple sequence variations unique to different populations, which could be explained by different evolutionary forces acting upon both domesticated rice. Our results highlight the interplay between crop domestication and cultivation and pathogen evolution.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB), a major rice disease causing up to 70% yield loss in Asia and West Africa. First described in Japan in 1884 and later reported in West Africa in the 1970s, BLB recently emerged in East Africa, with an epidemic reported in Tanzania in 2019. Remarkably, the disease was detected for the first time in Madagascar the same year, representing a serious threat to food security. To investigate the origin of BLB in Madagascar, we isolated 73 Xoo strains from symptomatic rice leaves collected between 2019 and 2023. Multilocus variable-number of tandem-repeats analysis genotyping revealed 19 haplotypes forming a single clonal complex, indicating low diversity and a likely recent introduction. To come up with disease control strategies, IRBB-based race-typing was achieved and identified four resistance genes (Xa8, xa13, Xa21, and Xa23) that confer resistance to all Malagasy strains tested, whereas the 19 Malagasy varieties assessed were susceptible. The analysis of SWEET knockout lines confirmed that Malagasy strains rely on the susceptibility gene OsSWEET11 for full virulence. Whole-genome sequencing and transcription activator-like effector repertoire analyses of two strains allowed for the identification of a PthXo1 ortholog predicted to induce OsSWEET11. Single-nucleotide polymorphism-based phylogenetic analyses clustered Malagasy strains within Asian lineages, most closely related to strains originated from India. Malagasy strains did not cluster with recently reported Tanzanian Xoo, suggesting independent introductions. Overall, our study demonstrates that BLB in Madagascar results from a recent and single introduction from Asia and identifies effective resistance genes for deployment. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Rice yellow mottle virus (RYMV) is one of the most devastating viral pathogens affecting rice cultivation in Africa. The eukaryotic translation initiation factor 4G isoform eIFiso4G1 plays a pivotal role in rice susceptibility to the virus. Naturally occuring resistance alleles impair infection but are predominantly found in the African cultivated species Oryza glaberrima, whose use in breeding programs is limited by interspecific sterility barriers with the widely grown O. sativa. Here, we used CRISPR-Cas9 to generate knockout (KO) mutations in eIFiso4G1 as well as insertion-deletion (Indel) variants within the region involved in interaction with the viral protein VPg. KO mutations conferred high resistance to RYMV. No resistance-breaking events were observed, suggesting that this resistance may be more durable than that conferred by naturally occuring alleles. However, complete gene KO slightly affected plant growth. Lines carrying Indel mutations in the VPg-interacting region displayed variable resistance levels, with some variants conferring high resistance without compromising plant development. Structural modeling of the eIFiso4G1 variants and their complexes with VPg provided mechanistic insights into how specific Indels modulate the resistance phenotype. ### Competing Interest Statement The authors have declared no competing interest. CGIAR, Research Program on rice agri-food systems (RICE, 2017–2022) IRD

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease of rice crops which can lead to yield losses ranging from 20 to 30% and as high as 50% (Mew 1987). BLB was first reported in Japan in 1884 and is now prevalent in most of the rice-producing countries in Asia. In Africa, Xoo was first reported in Mali and Cameroon (Buddenhagen et al., 1979) and later in many West African countries. Recently, BLB also appears to be spreading in East Africa with reports in Tanzania (Schepler-Luu et al., 2023) and in Madagascar (Raveloson et al., 2023). In June 2023, typical symptoms of BLB such as water-soaked areas and yellowish lesions along the leaf margins and tips were observed on rice plants in the coastal region of Kenya. Five rice fields of the Taita Taveta County with an incidence of 5-10% were surveyed, and 10-15 symptomatic leaf samples were collected in each field following a W pattern. Rice varieties were either Saro 5 or Arize 6444, two high productivity rice. 4-5cm long leaf fragments of 45 samples were next processed for bacterial isolation and surface disinfected, rinsed with sterile water, and ground individually in tubes with steel beads using a TissueLyser (Qiagen). Thirty-four round, pale yellow pigmented, and mucoid Xanthomonas-like colonies were obtained upon plating of 30 µl of the leaf powder resuspended in 1mL of sterile water on a semi-selective medium (peptone, 10 g/L; sucrose, 10 g/L; purified bacteriological agar, 16 g/L; glutamic acid, 1g/L; cycloheximide, 25 mg/L; cephalexin, 20 mg/L and kasugamycin, 10 mg/L) 72h after incubation at 28°C. To confirm that the isolates were Xoo, multiplex PCR established for the identification of X. oryzae pathovars was used (Lang et al. 2010). Thirty-two strains resulted in the amplification of two bands characteristic of the Xoo pattern. In addition, 6 of these strains (namely CIX5498, CIX5504, CIX5513, CIX5514, CIX5516 and CIX5539) were subjected to gyrB sequencing using the universal primers XgyrB1F and XgyrB1R (Young et al., 2008). The resulting partial 800 bp gyrB sequences of these 6 strains showed 100% identity (767/767 nt) with that of the PX099A Asian reference Xoo strain (accession CP000967.2). The pathogenicity of these 6 strains and the reference strain PX099A used as positive control was tested on 1-month-old Oryza sativa cv. Komboka plants (n=8 per treatment, 1 repetition). Rice leaves were inoculated by leaf clipping using sterile scissors dipped in the bacterial suspension at an optical density of 0.2. Three weeks after inoculation, plants exhibited typical BLB lesions for each of the six tested strains similarly to that of the positive control, whereas Mock-inoculated plants remained symptomless. Bacteria re-isolated from these diseased leaves yielded colonies that were confirmed as Xoo by multiplex PCR, fulfilling Koch's postulate. All strains isolated in this study are referenced in the CIX collection of the French National Research Institute for Sustainable Development in Montpellier. To our knowledge, this study represents the first molecular confirmation of the presence of BLB in Kenya and complement our knowledge of this important disease in East Africa. This work will serve as a basis for future rice breeding for BLB resistance efforts in Kenya. Further surveys are needed to clarify the spatial distribution and prevalence of BLB in other regions of Kenya and further analysis will be needed to understand the origin of the outbreak and to identify strategies to control BLB in Kenya.

Rice yellow mottle virus (RYMV) causes one of the most devastating rice diseases in Africa. Management of RYMV is challenging. Genetic resistance provides the most effective and environment-friendly control. The recessive resistance locus rymv2 (OsCPR5.1) had been identified in African rice (Oryza glaberrima), however, introgression into Oryza sativa ssp. japonica and indica remains challenging due to crossing barriers. Here, we evaluated whether CRISPR/Cas9 genome editing of the two rice nucleoporin paralogs OsCPR5.1 (RYMV2) and OsCPR5.2 can be used to introduce RYMV resistance into the japonica variety Kitaake. Both paralogs had been shown to complement the defects of the Arabidopsis atcpr5 mutant, indicating partial redundancy. Despite striking sequence and structural similarities between the two paralogs, only oscpr5.1 loss-of-function mutants were fully resistant, while loss-of-function oscpr5.2 mutants remained susceptible, intimating that OsCPR5.1 plays a specific role in RYMV susceptibility. Notably, edited lines with short in-frame deletions or replacements in the N-terminal domain (predicted to be unstructured) of OsCPR5.1 were hypersusceptible to RYMV. In contrast to mutations in the single Arabidopsis AtCPR5 gene, which caused severely dwarfed plants, oscpr5.1 and oscpr5.2 single and double knockout mutants showed neither substantial growth defects nor symptoms indicative lesion mimic phenotypes, possibly reflecting functional differentiation. The specific editing of OsCPR5.1, while maintaining OsCPR5.2 activity, provides a promising strategy for generating RYMV-resistance in elite Oryza sativa lines as well as for effective stacking with other RYMV resistance genes or other traits.

Bacterial leaf blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), threatens global food security and the livelihood of small-scale rice producers. Analyses of Xoo collections from Asia, Africa and the Americas demonstrated complete continental segregation, despite robust global rice trade. Here, we report unprecedented BB outbreaks in Tanzania. The causative strains, unlike endemic African Xoo, carry Asian-type TAL effectors targeting the sucrose transporter SWEET11a and iTALes suppressing Xa1. Phylogenomics clustered these strains with Xoo from Southern-China. African rice varieties do not carry effective resistance. To protect African rice production against this emerging threat, we developed a hybrid CRISPR-Cas9/Cpf1 system to edit all known TALe-binding elements in three SWEET promoters of the East African elite variety Komboka. The edited lines show broad-spectrum resistance against Asian and African strains of Xoo, including strains recently discovered in Tanzania. The strategy could help to protect global rice crops from BB pandemics.

SummaryRice yellow mottle virus (RYMV) causes one of the most devastating rice diseases in Africa. Management of RYMV is challenging. Genetic resistance provides the most effective and environment-friendly control. The recessive resistance locusrymv2(OsCPR5.1) had been identified in African rice (O. glaberrima), however, introgression intoO. sativa ssp. japonicaandindicaremains challenging due to crossing barriers. Here, we evaluated whether CRISPR/Cas9 genome editing of the two rice nucleoporin paralogsOsCPR5.1(RYMV2) andOsCPR5.2can be used to introduce RYMV resistance into thejaponicavariety Kitaake. Both paralogs had been shown to complement the defects of the Arabidopsisatcpr5mutant, indicating partial redundancy. Despite striking sequence and structural similarities between the two paralogs, only oscpr5.1loss-of-function mutants were fully resistant, while loss-of-functionoscpr5.2mutants remained susceptible, intimating thatOsCPR5.1plays a specific role in RYMV susceptibility. Notably, edited lines with short in-frame deletions or replacements in the N-terminal domain (predicted to be unstructured) ofOsCPR5.1were hypersusceptible to RYMV. In contrast to mutations in the single ArabidopsisAtCPR5gene, which caused severely dwarfed plants,oscpr5.1andoscpr5.2singleknockoutmutants show neither substantial growth defects nor symptoms indicative of programmed cell death, possibly reflecting functional redundancy of the isoforms regarding other important functions. The specific editing ofOsCPR5.1, while maintainingOsCPR5.2activity, provides a promising strategy for generating RYMV-resistance in eliteOryza sativalines as well as for effective stacking with other RYMV resistance genes or other traits.

Xanthomonas oryzae pv. oryzae (Xoo) strains that cause bacterial leaf blight (BLB) limit rice (Oryza sativa) production and require breeding more resistant varieties. Transcription activator-like effectors (TALEs) activate transcription to promote leaf colonization by binding to specific plant host DNA sequences termed effector binding elements (EBEs). Xoo major TALEs universally target susceptibility genes of the SWEET transporter family. TALE-unresponsive alleles of clade III OsSWEET susceptibility gene promoter created with genome editing confer broad resistance on Asian Xoo strains. African Xoo strains rely primarily on the major TALE TalC, which targets OsSWEET14. Although the virulence of a talC mutant strain is severely impaired, abrogating OsSWEET14 induction with genome editing does not confer equivalent resistance on African Xoo. To address this contradiction, we postulated the existence of a TalC target susceptibility gene redundant with OsSWEET14. Bioinformatics analysis identified a rice locus named ATAC composed of the INCREASED LEAF INCLINATION 2 (ILI2) gene and a putative lncRNA that are shown to be bidirectionally upregulated in a TalC-dependent fashion. Gain-of-function approaches with designer TALEs inducing ATAC sequences did not complement the virulence of a Xoo strain defective for SWEET gene activation. While editing the TalC EBE at the ATAC loci compromised TalC-mediated induction, multiplex edited lines with mutations at the OsSWEET14 and ATAC loci remained essentially susceptible to African Xoo strains. Overall, this work indicates that ATAC is a probable TalC off-target locus but nonetheless documents the first example of divergent transcription activation by a native TALE during infection.

The bacterial plant pathogen Xanthomonas oryzae pv. oryzae is responsible for the foliar rice bacterial blight disease. Genetically contrasted, continent-specific, sublineages of this species can cause important damages to rice production both in Asia and Africa. We report on the genome of the CIX2779 strain of this pathogen, previously named NAI1 and originating from Niger. Oxford Nanopore long reads assembly and Illumina short reads polishing produced a genome sequence composed of a 4,725,792-bp circular chromosome and a 39,798-bp-long circular plasmid designated pCIX2779_1. The chromosome structure and base-level sequence are highly related to reference strains of African X. oryzae pv. oryzae and encode identical transcription activator-like effectors for virulence. Importantly, our in silico analysis strongly indicates that pCIX2779_1 is a genuine conjugative plasmid, the first indigenous one sequenced from an African strain of the X. oryzae species. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

TBR225 is one of the most popular commercial rice varieties in Northern Vietnam. However, this variety is highly susceptible to bacterial leaf blight (BLB), a disease caused by Xanthomonas oryzae pv. oryzae (Xoo) which can lead to important yield losses. OsSWEET14 belongs to the SWEET gene family that encodes sugar transporters. Together with other Clade III members, it behaves as a susceptibility (S) gene whose induction by Asian Xoo Transcription-Activator-Like Effectors (TALEs) is absolutely necessary for disease. In this study, we sought to introduce BLB resistance in the TBR225 elite variety. First, two Vietnamese Xoo strains were shown to up-regulate OsSWEET14 upon TBR225 infection. To investigate if this induction is connected with disease susceptibility, nine TBR225 mutant lines with mutations in the AvrXa7, PthXo3 or TalF TALEs DNA target sequences of the OsSWEET14 promoter were obtained using the CRISPR/Cas9 editing system. Genotyping analysis of T0 and T1 individuals showed that mutations were stably inherited. None of the examined agronomic traits of three transgene-free T2 edited lines were significantly different from those of wild-type TBR225. Importantly, one of these T2 lines, harboring the largest homozygous 6-bp deletion, displayed decreased OsSWEET14 expression as well as a significantly reduced susceptibility to a Vietnamese Xoo strains and complete resistance to another one. Our findings indicate that CRISPR/Cas9 editing conferred an improved BLB resistance to a Vietnamese commercial elite rice variety.

Non-coding small RNAs (sRNA) act as mediators of gene silencing and regulate plant growth, development and stress responses. Early insights into plant sRNAs established a role in antiviral defense and they are now extensively studied across plant–microbe interactions. Here, sRNA sequencing discovered a class of sRNA in rice (Oryza sativa) specifically associated with foliar diseases caused by Xanthomonas oryzae bacteria. Xanthomonas-induced small RNAs (xisRNAs) loci were distinctively upregulated in response to diverse virulent strains at an early stage of infection producing a single duplex of 20–22 nt sRNAs. xisRNAs production was dependent on the Type III secretion system, a major bacterial virulence factor for host colonization. xisRNA loci overlap with annotated transcripts sequences, with about half of them encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and xisRNA loci predominantly coincide with the coding sequence of a conserved kinase motif. xisRNAs exhibit features of small interfering RNAs and their biosynthesis depend on canonical components OsDCL1 and OsHEN1. xisRNA induction possibly mediates post-transcriptional gene silencing but they do not broadly suppress cis-genes expression on the basis of mRNA-seq data. Overall, our results identify a group of unusual sRNAs with a potential role in plant–microbe interactions.

Bacterial blight of rice is an important disease in Asia and Africa. The pathogen, Xanthomonas oryzae pv. oryzae ( Xoo ), secretes one or more of six known transcription-activator-like effectors (TALes) that bind specific promoter sequences and induce, at minimum, one of the three host sucrose transporter genes SWEET11 , SWEET13 and SWEET14 , the expression of which is required for disease susceptibility. We used CRISPR–Cas9-mediated genome editing to introduce mutations in all three SWEET gene promoters. Editing was further informed by sequence analyses of TALe genes in 63 Xoo strains, which revealed multiple TALe variants for SWEET13 alleles. Mutations were also created in SWEET14 , which is also targeted by two TALes from an African Xoo lineage. A total of five promoter mutations were simultaneously introduced into the rice line Kitaake and the elite mega varieties IR64 and Ciherang-Sub1. Paddy trials showed that genome-edited SWEET promoters endow rice lines with robust, broad-spectrum resistance.

Rice bacterial leaf blight (BLB) is caused by Xanthomonas oryzae pv. oryzae (Xoo) which injects Transcription Activator-Like Effectors (TALEs) into the host cell to modulate the expression of target disease susceptibility genes. Xoo major-virulence TALEs universally target susceptibility genes of the SWEET sugar transporter family. TALE-unresponsive alleles of OsSWEET genes have been identified in the rice germplasm or created by genome editing and confer resistance to BLB. In recent years, BLB has become one of the major biotic constraints to rice cultivation in Mali. To inform the deployment of alternative sources of resistance in this country, rice lines carrying alleles of OsSWEET14 unresponsive to either TalF (formerly Tal5) or TalC, two important TALEs previously identified in West African Xoo, were challenged with a panel of strains recently isolated in Mali and were found to remain susceptible to these isolates. The characterization of TALE repertoires revealed that talF and talC specific molecular markers were simultaneously present in all surveyed Malian strains, suggesting that the corresponding TALEs are broadly deployed by Malian Xoo to redundantly target the OsSWEET14 gene promoter. Consistent with this, the capacity of most Malian Xoo to induce OsSWEET14 was unaffected by either talC- or talF-unresponsive alleles of this gene. Long-read sequencing and assembly of eight Malian Xoo genomes confirmed the widespread occurrence of active TalF and TalC variants and provided a detailed insight into the diversity of TALE repertoires. All sequenced strains shared nine evolutionary related tal effector genes. Notably, a new TalF variant that is unable to induce OsSWEET14 was identified. Furthermore, two distinct TalB variants were shown to have lost the ability to simultaneously induce two susceptibility genes as previously reported for the founding members of this group from strains MAI1 and BAI3. Yet, both new TalB variants retained the ability to induce one or the other of the two susceptibility genes. These results reveal molecular and functional differences in tal repertoires and will be important for the sustainable deployment of broad-spectrum and durable resistance to BLB in West Africa.

SummaryAs a key virulence strategy to cause bacterial leaf blight, Xanthomonas oryzae pv. oryzae (Xoo) injects into the plant cell DNA‐binding proteins called transcription activator‐like effectors (TALEs) that bind to effector‐binding elements (EBEs) in a sequence‐specific manner, resulting in host gene induction. TALEs AvrXa7, PthXo3, TalC and Tal5, found in geographically distant Xoo strains, all target OsSWEET14, thus considered as a pivotal TALE target acting as major susceptibility factor during rice–Xoo interactions. Here, we report the generation of an allele library of the OsSWEET14 promoter through stable expression of TALE‐nuclease (TALEN) constructs in rice. The susceptibility level of lines carrying mutations in AvrXa7, Tal5 or TalC EBEs was assessed. Plants edited in AvrXa7 or Tal5 EBEs were resistant to bacterial strains relying on the corresponding TALE. Surprisingly, although indels within TalC EBE prevented OsSWEET14 induction in response to BAI3 wild‐type bacteria relying on TalC, loss of TalC responsiveness failed to confer resistance to this strain. The TalC EBE mutant line was, however, resistant to a strain expressing an artificial SWEET14‐inducing TALE whose EBE was also edited in this line. This work offers the first set of alleles edited in TalC EBE and uncovers a distinct, broader range of activities for TalC compared to AvrXa7 or Tal5. We propose the existence of additional targets for TalC beyond SWEET14, suggesting that TALE‐mediated plant susceptibility may result from induction of several, genetically redundant, host susceptibility genes by a single effector.

Hirschfeldia incana, a pseudometallophyte belonging to the Brassicaceae family and widespread in the Mediterranean region, was selected for its ability to grow on soils contaminated by lead (Pb). The global comparison of gene expression using microarrays between a plant susceptible to Pb (Arabidopsis thaliana) and a Pb tolerant plant (H. incana) enabled the identification of a set of specific genes expressed in response to lead exposure. Three groups of genes were particularly over-represented by the Pb exposure in the biological processes categorized as photosynthesis, cell wall, and metal handling. Each of these gene groups was shown to be directly involved in tolerance or in protection mechanisms to the phytotoxicity associated with Pb. Among these genes, we demonstrated that MT2b, a metallothionein gene, was involved in lead accumulation, confirming the important role of metallothioneins in the accumulation and the distribution of Pb in leaves. On the other hand, several genes involved in biosynthesis of ABA were shown to be up-regulated in the roots and shoots of H. incana treated with Pb, suggesting that ABA-mediated signaling is a possible mechanism in response to Pb treatment in H. incana. This latest finding is an important research direction for future studies.

Root nodule symbioses (RNS) allow plants to acquire atmospheric nitrogen by establishing an intimate relationship with either rhizobia, the symbionts of legumes or Frankia in the case of actinorhizal plants. In legumes, NIN (Nodule INception) genes encode key transcription factors involved in nodulation. Here we report the characterization of CgNIN, a NIN gene from the actinorhizal tree Casuarina glauca using both phylogenetic analysis and transgenic plants expressing either ProCgNIN::reporter gene fusions or CgNIN RNAi constructs. We have found that CgNIN belongs to the same phylogenetic group as other symbiotic NIN genes and CgNIN is able to complement a legume nin mutant for the early steps of nodule development. CgNIN expression is correlated with infection by Frankia, including preinfection stages in developing root hairs, and is induced by culture supernatants. Knockdown mutants were impaired for nodulation and early root hair deformation responses were severely affected. However, no mycorrhizal phenotype was observed and no induction of CgNIN expression was detected in mycorrhizas. Our results indicate that elements specifically required for nodulation include NIN and possibly related gene networks derived from the nitrate signalling pathways.

Lead is a heavy metal of particular concern with respect to environmental quality and health. The lack of plant species that accumulate and tolerate Pb is a limiting factor to understand the molecular mechanisms involved in Pb tolerance. In this study we identified Hirschfeldia incana, a Brassicaceae collected from metalliferous mine spoils in Morocco, as a Pb accumulator plant. H. incana exhibited high Pb accumulation in mine soils and in hydroponic cultures. Major Pb accumulation occurred in the roots and a part of Pb translocated from the roots to the shoots, even to the siliques. These findings demonstrated that H. incana is a Pb accumulator species. The expression of several candidate genes after Pb-exposure was measured by quantitative PCR and two of them, HiHMA4 and HiMT2a, coding respectively for a P1B-type ATPase and a metallothionein, were particularly induced by Pb-exposure in both roots and leaves. The functional characterization of HiHMA4 and HiMT2a was achieved using Arabidopsis T-DNA insertional mutants. Pb content and primary root growth analysis confirmed the role of these two genes in Pb tolerance and accumulation. H. incana could be considered as a good experimental model to identify genes involved in lead tolerance and accumulation in plants.