Marker-assisted improvement of the elite restorer line of rice,RPHR-1005 for resistance against bacterial blight and blast diseases

This study was carried out to improve the RPHR-1005, a stable restorer line of the popular medium slender grain type rice hybrid, DRRH-3 for bacterial blight (BB) and blast resistance through marker-assisted backcross breeding (MABB). Two major BB resistance genes, Xa21 and Xa33 and a major blast resistance gene, Pi2 were transferred to RPHR-1005 as two individual crosses. Foreground selection for Xa21, Xa33, Pi2, Rf3 and Rf4 was done by using gene-specific functional markers, while 59 simple sequence repeat (SSR) markers polymorphic between the donors and recipient parents were used to select the best plant possessing target resistance genes at each backcross generation. Backcrossing was continued till BC ₂ F ₂ and a promising homozygous backcross derived line possessing Xa21 + Pi2 and another possessing Xa33 were intercrossed to stack the target resistance genes into the genetic background of RPHR-1005. At ICF ₄, 10 promising lines possessing three resistance genes in homozygous condition along with fine-grain type, complete fertility restoration, better panicle exertion and taller plant type (compared to RPHR-1005) were identified.     

Marker-assisted introgression of the major bacterial blight resistance gene, <Emphasis Type="Italic">Xa21</Emphasis> and blast resistance gene, <Emphasis Type="Italic">Pi54</Emphasis> into RPHR-1005, the restorer line of the popular rice hybrid,DRRH3

The elite Indian rice hybrid, DRRH3 is highly susceptible to two major diseases, bacterial blight (BB) and blast, which limit its productivity significantly. In the present study, we have introgressed two major genes, viz., Xa21 and Pi54 conferring resistance against BB and blast, respectively into RPHR-1005, the male parent of DRRH3 through marker-assisted backcross breeding (MABB) and analyzed the backcross derived plants for their resistance against BB and blast. RPBio Patho-2 was used as a donor for both the resistance genes. Gene-specific markers were used for the foreground selection of Xa21 and Pi54 at each stage of backcrossing and markers specific for the major fertility restorer genes, Rf3 and Rf4 were used only at BC₁F₁ generation for foreground selection. Background selection was done using 62 polymorphic SSR markers and marker-assisted backcrossing was continued till BC₃ generation. At BC₃F₄, through intensive phenotype-based selections 15 promising lines (ABLs) possessing high level of resistance against BB and blast, high yield, fine-grain type, complete fertility restoration along with better panicle exsertion and taller plant type as compared to RPHR-1005 were identified and test crossed with APMS 6 A, the female parent of DRRH3. The newly derived hybrids (i.e. improved versions of DRRH3) were observed to possess high level of resistance against BB and blast along with medium-slender grain type and yield level better than or equivalent to that of DRRH3. Our study exemplifies the utility of MABB for targeted improvement of multiple traits in hybrid rice.     

Improved versions of rice maintainer line,APMS 6B,possessing two resistance genes, <Emphasis Type="Italic">Xa21</Emphasis> and <Emphasis Type="Italic">Xa38</Emphasis>, exhibit high level of resistance to bacterial blight disease

APMS 6B is the stable maintainer of the CMS line APMS 6A, which is the female parent of the popular Indian rice hybrid DRRH 3. APMS 6B has good combining ability and plant stature but is highly susceptible to bacterial blight (BB) disease. In order to improve the BB resistance of APMS 6B, we pyramided two major, dominant BB resistance genes, Xa21 and Xa38, through marker-assisted backcross breeding (MABB). Improved Samba Mahsuri (ISM) was used as the donor for Xa21 while PR 114 (Xa38) served as the donor for Xa38. Individual crosses [APMS 6B/ISM and APMS 6B/PR 114 (Xa38)] were performed, and true F₁ plants were then backcrossed with APMS 6B and the MABB process was continued till BC₃. A single positive BC₃F₁ plant identified from both the crosses with maximum genotypic and phenotypic similarity with APMS 6B was selfed to generate BC₃F₂s. At BC₃F₂ generation, plants homozygous for either Xa21 or Xa38 were identified and further confirmed for the absence of two major fertility restorer genes, Rf3 and Rf4. A single such homozygous BC₃F₂ plant, each from both the crosses, was then inter-mated to generate ICF₁s (inter-cross F₁s). Selected ICF₁ plants possessing both the BB resistance genes were selfed to generate ICF₂s. A total of 42 ICF₂ plants homozygous for both Xa21 and Xa38 were identified and screened with parental polymorphic SSR markers to identify the best F₂ plants having the maximum recurrent parent genome recovery. Twelve best ICF₂ plants were advanced up to ICF₅. The ICF₅ lines displayed very high level of BB resistance and were similar to APMS 6B in terms of agro-morphological characters. Further, most of these lines also showed complete maintenance ability and such lines are being advanced for conversion to WA-CMS lines.     

Improvement of blast resistance of the popular high-yielding,medium slender-grain type,bacterial blight resistant rice variety,Improved Samba Mahsuri by marker-assisted breeding

Improved Samba Mahsuri (ISM) is a popular, high-yielding, bacterial blight resistant rice variety possessing medium-slender grain type. As ISM is highly susceptible to blast disease of rice, through the present study we have transferred two major blast resistance genes, Pi2 and Pi54 into the elite variety by marker-assisted backcross breeding. The two blast resistance genes were transferred to ISM through sets of backcrosses. In every backcross generation, PCR-based markers, specific for the blast resistance genes (Pi2 and Pi54) and bacterial blight resistance genes (Xa21, xa13 and xa5) were utilized for foreground selection, while a set of 144 parental polymorphic SSR markers were used for background selection and backcrossing was carried out until BC₂ generation. A solitary BC₂F₁ plant possessing Pi2 or Pi54 along with Xa21, xa13 and xa5 and >?90% recovery of ISM genome was selected from the two sets of backcrosses were crossed and the intercross F₁s (ICF₁s) thus obtained were selfed to generate ICF₂s. Homozygous ICF₂ plants carrying all the five resistance genes were identified through markers and advanced through selfing till ICF₅ generation by adopting pedigree method of selection. Three best lines at ICF₅, possessing excellent resistance against bacterial blight and blast and closely resembling or superior to ISM in terms of grain quality: yield and agro-morphological traits have been identified and advanced for multi-location trials.     

Exploring the mechanism and efficient use of a durable gene-mediated resistance to bacterial blight disease in rice

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, the most devastating bacterial disease of rice worldwide. The major disease resistance gene Xa3/Xa26 confers a durable resistance to Xoo with a dosage effect. However, the mechanism of Xa3/Xa26-mediated resistance remains to be elucidated. We created near-isogenic lines carrying Xa3/Xa26 with a background of indica and japonica, the two major subspecies of Asian cultivated rice. Analyzing these rice lines showed that the japonica background facilitated resistance to Xoo, which was associated with increased Xa3/Xa26 expression, compared with rice lines with an indica background. This characteristic of Xa3/Xa26 was related to the WRKY45 locus, which had higher expression with the japonica background than with the indica background. However, the two alleles of the WRKY45 locus had different expression levels, with the WRKY45-1 expression level being higher than that of WRKY45-2 for both japonica and indica backgrounds. In addition, the resistance level conferred by Xa3/Xa26 was higher in the presence of WRKY45-1 than in the presence of WRKY45-2 for both japonica and indica backgrounds. Xa3/Xa26-mediated resistance was associated with increased accumulation of jasmonic acid (JA), JA-isoleucine, and terpenoid and flavonoid phytoalexins. Exogenous JA application enhanced Xa3/Xa26-mediated resistance. These results not only provide more knowledge toward understanding the mechanism of Xa3/Xa26-mediated resistance but also offer the best choice for using Xa3/Xa26 for rice resistance improvement, specifically, a japonica background with the WRKY45-1 allele.     

Analysis of SSH library of rice variety Aganni reveals candidate gall midge resistance genes

The Asian rice gall midge, Orseolia oryzae, is a serious insect pest causing extensive yield loss. Interaction between the gall midge and rice genotypes is known to be on a gene-for-gene basis. Here, we report molecular basis of HR? (hypersensitive reaction—negative) type of resistance in Aganni (an indica rice variety possessing gall midge resistance gene Gm8) through the construction and analysis of a suppressive subtraction hybridization (SSH) cDNA library. In all, 2,800 positive clones were sequenced and analyzed. The high-quality ESTs were assembled into 448 non-redundant gene sequences. Homology search with the NCBI databases, using BlastX and BlastN, revealed that 73% of the clones showed homology to genes with known function and majority of ESTs belonged to the gene ontology category ‘biological process’. Validation of 27 putative candidate gall midge resistance genes through real-time PCR, following gall midge infestation, in contrasting parents and their derived pre-NILs (near isogenic lines) revealed induction of specific genes related to defense and metabolism. Interestingly, four genes, belonging to families of leucine-rich repeat (LRR), heat shock protein (HSP), pathogenesis related protein (PR), and NAC domain-containing protein, implicated in conferring HR+ type of resistance, were found to be up-regulated in Aganni. Two of the reactive oxygen intermediates (ROI)–scavenging-enzyme-coding genes Cytosolic Ascorbate Peroxidase1, 2 (OsAPx1 and OsAPx2) were found up-regulated in Aganni in incompatible interaction possibly suppressing HR. We suggest that Aganni has a deviant form of inducible, salicylic acid (SA)-mediated resistance but without HR.     

Identification of novel recessive gene <Emphasis Type="Italic">xa44</Emphasis>(<Emphasis Type="Italic">t</Emphasis>) conferring resistance to bacterial blight races in rice by QTL linkage analysis using an SNP chip

Key message

Using QTL analysis and fine mapping, the novel recessive gene xa44(t) conferring resistance to BB was identified and the expression level of the gene was confirmed through qRT-PCR analysis.

Abstract

Bacterial blight (BB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major factor causing rice yield loss in most rice-cultivating countries, especially in Asia. The deployment of cultivars with resistance to BB is the most effective method to control the disease. However, the evolution of new Xoo or pathotypes altered by single-gene-dependent mutations often results in breakdown of resistance. Thus, efforts to identify novel R-genes with sustainable BB resistance are urgently needed. In this study, we identified three quantitative trait loci (QTLs) on chromosomes 1, 4, and 11, from an F₂ population of 493 individuals derived from a cross between IR73571-3B-11-3-K3 and Ilpum using a 7K SNP chip. Of these QTLs, one major QTL, qBB_11, on chromosome 11 explained 61.58% of the total phenotypic variance in the population, with an LOD value of 113.59, based on SNPs 11964077 and 11985463. The single major R-gene, with recessive gene action, was designated xa44(t) and was narrowed down to a 120-kb segment flanked within 28.00 Mbp to 28.12 Mbp. Of nine ORFs present in the target region, two ORFs revealed significantly different expression levels of the candidate genes. These candidate genes (Os11g0690066 and Os11g0690466) are described as “serine/threonine protein kinase domain containing protein” and “hypothetical protein,” respectively. The results will be useful to further understand BB resistance mechanisms and provide new sources of resistance, together with DNA markers for MAS breeding to improve BB resistance in rice.

     

The Receptor-Like Cytoplasmic Kinase OsRLCK102 Regulates XA21-Mediated Immunity and Plant Development in Rice

Receptor-like cytoplasmic kinases (RLCKs) belong to a large subgroup of kinases that play pivotal roles in plant development and in protecting plants from various stresses. Here, we report the isolation and characterization of rice OsRLCK102, from the OsRLCK VII subgroup. Silencing of OsRLCK102 compromised receptor kinase XA21-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo) but did not affect plant basal resistance to Xoo or Magnaporthe oryzae (M. oryzae). Plants with silenced OsRLCK102 exhibit architecture alterations, including reduced plant height, enlarged angle of the lamina joint, decreased rates of seed setting and enhanced sensitivity to hormone brassinolide (BR). Collectively, our study reveals that OsRLCK102 positively regulates XA21-mediated immunity and negatively regulates rice development through BR signaling in rice.     

Identification of a specific molecular marker for the rice blast-resistant gene <Emphasis Type="Italic">Pigm</Emphasis> and molecular breeding of thermo-sensitive genic male sterile leaf-color marker lines

Rice blast is a damaging disease caused by Magnaportheoryzae. Marker-assisted selection of blast resistance genes could help develop cultivars with blast resistance. Pigm is a broad-spectrum blast-resistant gene. However, few rice resources contain Pigm. In this study, the Pigm gene donor Gumei4 (GM4) was investigated. By analyzing different regions of Pigm sequences, we found that marker G8900 was a specific molecular marker of Pigm gene in GM4. Correlation analysis between molecular marker detection and identification of rice blast disease nursery revealed that G8900 could be used in marker-assisted selection (MAS) of Pigm. Furthermore, we introduced Pigm gene into the KT27S line (a blast-susceptible yellow-green-leaf-color mutant) in G8900-assisted breeding and identified three new yellow-green-leaf-color marker lines that are resistant to blast. The agronomic and economic traits of the three new lines are similar to those of their parental lines. The identification and application of Pigm-specific molecular marker in breeding of yellow-green-leaf-color marker line could play an important role in the production of disease-resistant hybrid rice.     

Marker-assisted breeding of Chinese elite rice cultivar 9311 for disease resistance to rice blast and bacterial blight and tolerance to submergence

Rice (Oryza sativa L.) is the staple food crop for more than half of the world’s population. The development of hybrid rice is a practical approach to increase rice production. However, rice production was frequently affected by biotic and abiotic stresses. Rice blast and bacterial blight are two major diseases in rice growing regions. Rice plantation is also frequently affected by short-term submergence or seasonal floods in wet seasons and drought in dry seasons. The utilization of natural disease resistance (R) genes and stress tolerance genes in rice breeding is the most economic and efficient way to combat or adapt to these biotic and abiotic stresses. Rice cultivar 9311 is widely planted rice variety, either as inbred rice or the paternal line of two-line hybrid rice. Here, we report the pyramiding of rice blast R gene Pi9, bacterial blight R genes Xa21 and Xa27, and submergence tolerance gene Sub1A in 9311 genetic background through backcrossing and marker-assisted selection. The improved rice line, designated as 49311, theoretically possesses 99.2% genetic background of 9311. 49311 and its hybrid rice, GZ63S/49311, conferred disease resistance to rice blast and bacterial blight and showed tolerance to submergence for over 18 days without significant loss of viability. 49311 and its hybrids had similar agronomic traits and grain quality to 9311 and the control hybrid rice, respectively. The development of 49311 provides an improved paternal line for two-line hybrid rice production with disease resistance to rice blast and bacterial blight and tolerance to submergence.     

Improvement of seedling and panicle blast resistance in <Emphasis Type="Italic">Xian</Emphasis> rice varieties following <Emphasis Type="Italic">Pish</Emphasis> introgression

Rice blast is a serious disease caused by the filamentous ascomycetous fungus Magnaporthe oryzae. Incorporating disease resistance genes in rice varieties and characterizing the distribution of M. oryzae isolates form the foundation for enhancing rice blast resistance. In this study, the blast resistance gene Pish was observed to be differentially distributed in the genomes of rice sub-species. Specifically, Pish was present in 80.5% of Geng varieties, but in only 2.3% of Xian varieties. Moreover, Pish conferred resistance against only 23.5% of the M. oryzae isolates from the Geng-planting regions, but against up to 63.2% of the isolates from the Xian-planting regions. Thus, Pish may be an elite resistance gene for improving rice blast resistance in Xian varieties. Therefore, near-isogenic lines (NILs) with Pish and the polygene pyramid lines (PPLs) PPL^Pish/Pi1, PPL^Pish/Pi54, and PPL^Pish/Pi33 in the Xian background Yangdao 6 were generated using a molecular marker-assisted selection method. The results suggested that (1) Pish significantly improved rice blast resistance in Xian varieties, which exhibited considerably improved seedling and panicle blast resistance after Pish was introduced; (2) PPLs with Pish were more effective than the NILs with Pish regarding seedling and panicle blast resistance; (3) the PPL seedling and panicle blast resistance was improved by the complementary and overlapping effects of different resistance genes; and (4) the stability of NIL and PPL resistance varied under different environmental conditions, with only PPL^Pish/Pi54 exhibiting highly stable resistance in three natural disease nurseries (Jianyang, Jinggangshan, and Huangshan). This study provides new blast resistance germplasm resources and describes a novel molecular strategy for enhancing rice blast resistance.     

Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice

Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. ‘W. Murcott’ mandarin (a hybrid of ‘Murcott’ and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of ‘W. Murcott’ mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of ‘W. Murcott’ mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3–5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic ‘W. Murcott’ mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.     

Nitrogen Metabolism Genes from Temperate Marine Sediments

In this study, we analysed metagenomes along with biogeochemical profiles from Skagerrak (SK) and Bothnian Bay (BB) sediments, to trace the prevailing nitrogen pathways. NO₃ ^? was present in the top 5 cm below the sediment-water interface at both sites. NH₄ ⁺ increased with depth below 5 cm where it overlapped with the NO₃ ^? zone. Steady-state modelling of NO₃ ^? and NH₄ ⁺ porewater profiles indicates zones of net nitrogen species transformations. Bacterial protease and hydratase genes appeared to make up the bulk of total ammonification genes. Genes involved in ammonia oxidation (amo, hao), denitrification (nir, nor), dissimilatory NO₃ ^? reduction to NH₄ ⁺ (nfr and otr) and in both of the latter two pathways (nar, nap) were also present. Results show ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) are similarly abundant in both sediments. Also, denitrification genes appeared more abundant than DNRA genes. 16S rRNA gene analysis showed that the relative abundance of the nitrifying group Nitrosopumilales and other groups involved in nitrification and denitrification (Nitrobacter, Nitrosomonas, Nitrospira, Nitrosococcus and Nitrosomonas) appeared less abundant in SK sediments compared to BB sediments. Beggiatoa and Thiothrix 16S rRNA genes were also present, suggesting chemolithoautotrophic NO₃ ^? reduction to NO₂ ^? or NH₄ ⁺ as a possible pathway. Our results show the metabolic potential for ammonification, nitrification, DNRA and denitrification activities in North Sea and Baltic Sea sediments.     

Genome-Wide Analysis in Wild and Cultivated <Emphasis Type="Italic">Oryza</Emphasis> Species Reveals Abundance of NBS Genes in Progenitors of Cultivated Rice

NBS-encoding genes play a critical role in the plant defense system. Wild relatives of crop plants are rich reservoirs of plant defense genes. Here, we performed a stringent genome-wide identification of NBS-encoding genes in three cultivated and eight wild Oryza species, representing three different genomes (AA, BB, and FF) from four continents. A total of 2688 NBS-encoding genes were identified from 11 Oryza genomes. All the three progenitor species of cultivated rice, namely O. barthii, O. rufipogon, and O. nivara, were the richest reservoir of NBS-encoding genes (214, 313, and 307 respectively). Interestingly, the two Asian cultivated species showed a contrasting pattern in the number of NBS-encoding genes. While indica subspecies maintained nearly equal number of NBS genes as its progenitor (309 and 313), the japonica subspecies had retained only two third in the course of evolution (213 and 307). Other major sources for NBS-encoding genes could be (i) O. longistaminata since it had the highest proportion of NBS-encoding genes and (ii) O. glumaepatula as it clustered distinctly away from the rest of the AA genome species. The present study thus revealed that NBS-encoding genes can be exploited from the primary gene pool for disease resistance breeding in rice.     

Characterization of harpin<Subscript>Xoo</Subscript> induced hypersensitive responses in non host plant,tobacco

Harpin proteins encoded by hrp genes are bacterial protein elicitors that can stimulate hypersensitive response (HR) in non-host plants. HR-related pathogen resistance involves a complex form of programmed cell death (PCD). It is increasingly viewed as a key component of the hypersensitive disease response of plants. Currently, the evidence of harpin proteins-induced PCD is deficient though it exhibits phenotypic parallels with HR, and the mechanism of harpin proteins-induced PCD is not well understood. In this study, we demonstrate that harpin_Xoo protein from Xanthomonas oryzae pv. oryzae of rice bacterial blight expressed and isolated from bacterial cells acted as an agent to induce PCD in infiltrated tobacco plants. Treatment of tobacco leaves with harpin_Xoo induced typical PCD-related morphological and biochemical changes including cell shrinkage and nuclear DNA degradation. We further analyzed the expression of several genes in signal transduction pathway of PCD in tobacco plants by real-time qRT-PCR analysis using EF-1α as an endogenous control. Our results showed that the expression of NtDAD1 was down-regulated and the expression of BI-1, tpa1 and aox1 was up-regulated following the infiltration of harpin_Xoo into tobacco leaves. Our data suggest that harpin_Xoo can induce PCD with the coordination of PCD-related genes in infiltrated tobacco leaves, providing evidence to further investigate the signal transduction pathways of HR and PCD.     

Development and validation of candidate gene-specific markers for the major fertility restorer genes, <Emphasis Type="Italic">Rf4</Emphasis> and <Emphasis Type="Italic">Rf3</Emphasis> in rice

Two major nuclear genes, Rf3 and Rf4, are known to be associated with fertility restoration of wild-abortive cytoplasmic male sterility (WA-CMS) in rice. In the present study, through a comparative sequence analysis of the reported putative candidate genes, viz. PPR9-782-(M,I) and PPR762 (for Rf4) and SF21 (for Rf3), among restorer and maintainer lines of rice, we identified significant polymorphism between the two lines and developed a set of PCR-based codominant markers, which could distinguish maintainers from restorers. Among the five markers developed targeting the polymorphisms in PPR9-782-(M,I), the marker RMS-PPR9-1 was observed to show clear polymorphism between the restorer (n = 120) and maintainer lines (n = 44) analyzed. Another codominant marker, named RMS-PPR762 targeting PPR762, displayed a lower efficiency in identification of restorers and maintainers, indicating that PPR9-782-(M,I) is indeed the candidate gene for Rf4. With respect to Rf3, a codominant marker, named RMS-SF21-5 developed targeting SF21, displayed significantly lower efficiency in identification of restorers and non-restorers as compared to the Rf4-specific markers. Validation of these markers in a F₂ mapping population segregating for fertility restoration indicated that Rf4 has a major influence on fertility restoration and Rf3 is a minor gene. Further, the functional marker RMS-PPR9-1 was observed to be very useful in identification of impurities in a seed lot of the popular hybrid, DRRH3. Interestingly, when RMS-PPR9-1 and RMS-SF21-5 were considered in conjunction with analysis, near-complete, marker–trait co-segregation was observed, indicating that deployment of the candidate gene-specific markers both Rf4 and Rf3, together, can be helpful in accurate identification of fertility restorer lines and can facilitate targeted transfer of the two restorer genes into elite varieties through marker-assisted breeding.     

First study on characterization of virulence and antibiotic resistance genes in verotoxigenic and enterotoxigenic <Emphasis Type="Italic">E</Emphasis>. <Emphasis Type="Italic">coli</Emphasis> isolated from raw milk and unpasteurized traditional cheeses in Romania

The study focused on the incidence of enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) in raw milk and traditional dairy cheeses marketed in Romania, characterizing the virulence and antibiotic resistance genes of these isolates. One hundred and twenty samples of raw milk and 80 samples of unpasteurized telemy cheese were collected and cultured according to the international standard protocol. All the characteristic E. coli cultures were analyzed for the presence of STa, STb, LT, stx1, and stx2 toxicity genes. The ETEC/VTEC strains were tested for the presence of antibiotic resistance genes, such as aadA1, tetA, tetB, tetC, tetG, dfrA1, qnrA, aaC, sul1, bla _SHV , bla _CMY , bla _TEM , and ere(A), using PCR. The results showed that 27 samples (18.62%) were positive for one of the virulence genes investigated. 48.1% (n = 13) tested positive at the genes encoding for tetracycline resistance, tetA being the most prevalent one (61.5%; n = 8). A high percent (33.3%; n = 9) revealed the beta-lactamase (bla _TEM ) resistance gene, and none of the samples tested positive for bla _CMY and bla _SHV genes. The genes responsible for resistance to sulfonamides (sul1) and trimethoprim (dfrA1) were detected in rates of 14.8% (n = 4) and 7.4% (n = 2), respectively. E. coli is highly prevalent in raw milk and unpasteurized cheeses marketed in Romania. These strains might represent an important reservoir of resistance genes which can easily spread into other European countries, given the unique market.     

Molecular characterization and subcellular localization of salt-inducible lipid transfer proteins in rice

Rice (Oryza sativa L.) is a salt-sensitive species. Salt stress can cause injury to the plant cellular membrane. Plant lipid transfer proteins (LTPs) are abundant lipid binding proteins that are important in membrane vesicle biogenesis and trafficking, however, the biological importance of LTPs on salt-stress response in rice remains unclear. Therefore, salt-responsive rice LTPs were identified and characterized in this study. Microarray analysis showed seven genes positively regulated by salinity, including five Ltp genes (LtpII.3, LtpII.5, LtpII.6, LtpV.1, and LtpV.2) and two Ltp-like (LtpL; LtpL1, and LtpL2) genes. Amino acid alignment revealed that all these Ltp and LtpL genes contained the N-terminal signal peptide. Apart from LtpL1, all salt-inducible Ltp genes had the conserved eight cysteine residue motifs backbone. Verification of gene expression to different stimuli in rice seedlings revealed that salt-regulated Ltp genes differentially responded to drought, cold, H₂O₂, abscisic acid (ABA) and CaCl₂. Furthermore, the expression of Ltp and LtpL genes was tissue-specifically regulated by ABA-dependent and independent pathway. In silico analysis of a 1.5-kb 5’-upstream region of these genes showed regulatory cis-elements associated with ABA, calcium, and cold/drought responses. Three LtpII subfamily genes, including LtpII.3, LtpII.5, and LtpII.6, were strictly expressed in flowers and seeds, and LtpIII.1 mRNA strongly accumulated in stem tissue. Subcellular localization analysis of LTP-DsRed fusion proteins revealed that the five LTPs and two LTPLs localized at the endoplasmic reticulum. The results provide new clues to further understanding the biological functions of Ltp genes.