Resistant Near-isogenic Lines Research Articles

Transcriptome analysis revealed differentially expressed genes in rice functionally associated with brown planthopper defense in near isogenic lines pyramiding BPH14 and BPH15.

Although rice has many pests, brown planthopper (BPH) in particular is known to cause substantial damage. The pyramiding application of BPH-resistance genes BPH14 and BPH15 has proven effective in enhancing rice defense against BPH. However, the molecular mechanisms underlying BPH14/BPH15-conferred resistance remain unexplained. In this investigation, we analyzed the transcriptomes of near isogenic lines (NILs) containing either BPH14 (B14), BPH15 (B15), or BPH14/BPH15 (B1415), as well as their recurrent parent (RP) 'Wushansimiao'. In total, we detected 14,492 differentially expressed genes (DEGs) across 12 mRNA profiles of resistant NILs and RP at different feeding stages. In the transcriptomic analysis, 531 DEGs appeared to be common among the resistant NILs compared to RP before and after BPH feeding. These common DEGs were enriched in defense response, phosphorylation, and salt stress response. In addition, 258 DEGs shared only in resistant NILs were obtained among the different feeding stages, which were enriched in oxidative stress response, karrikin response, and chloroplast organization. Considering the expression patterns and relevant research reports associated with these DEGs, 21 were chosen as BPH resistance candidates. In rice protoplasts, the candidate DEG OsPOX8.1 was confirmed to increase reactive oxygen species (ROS) accumulation by chemiluminescence measurement. Our results provide valuable information to further explore the defense mechanism of insect-resistant gene pyramiding lines and develop robust strategies for insect control.

Determining the yield performance of near isogenic lines (NILs) in maize

The present experiment was conducted at Department of Millets, Centre for Plant breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India to analysis the yield performance of sorghum down mildew resistant near isogenic lines in Maize.The objective of this study was to assess the yield performance of four sorghum downy mildew resistant NILs in maize. Four SDM resistant NILs viz., 79/936- C1-7-7-7-46-2, 79/936- C1-7-7-7-80-17, 79/936- C1-7-7-7-92-1 and 79/936- C1- 7-7-7-92-7 were used for this purpose. Nine biometrical characters viz.,days to 50% oftasseling, days to 50% of silking, Days to maturity, Plant height , Ear height, Cob length, Cob diameter, 100 grain weight and yield per plant were taken for analysis . The study revealed that all the four NILs showed better per se performance for yield contributing characters and more mean values than the parents hence, these four Near Isogenic Lines (NILs) may be used as a parent to develop hybrid in maize breeding programme.

Gene B5 in Cotton Confers High and Broad Resistance to Bacterial Blight and Conditions High Amounts of Sesquiterpenoid Phytoalexins.

Bacterial blight resistance gene B5 has received little attention since it was first described in 1950. A near-isogenic line (NIL) of Gossypium hirsutum cotton, AcB5, was generated in an otherwise bacterial-blight-susceptible 'Acala 44' background. The introgressed locus B5 in AcB5 conferred strong and broad-spectrum resistance to bacterial blight. Segregation patterns of test crosses under Oklahoma field conditions indicated that AcB5 is likely homozygous for resistance at two loci with partial dominance gene action. In controlled-environment conditions, two of the four copies of B5 were required for effective resistance. Contrary to expectations of gene-for-gene theory, AcB5 conferred high resistance toward isogenic strains of Xanthomonas citri subsp. malvacearum carrying cloned avirulence genes avrB4, avrb7, avrBIn, avrB101, and avrB102, respectively, and weaker resistance toward the strain carrying cloned avrb6. The hypothesis that each B gene, in the absence of a polygenic complex, triggers sesquiterpenoid phytoalexin production was tested by measurement of cadalene and lacinilene phytoalexins during resistant responses in five NILs carrying different B genes, four other lines carrying multiple resistance genes, as well as susceptible Ac44E. Phytoalexin production was an obvious, but variable, response in all nine resistant lines. AcB5 accumulated an order of magnitude more of all four phytoalexins than any of the other resistant NILs. Its total levels were comparable to those detected in OK1.2, a highly resistant line that possesses several B genes in a polygenic background.

Comparative transcriptome-wide identification and differential expression of genes and lncRNAs in rice near-isogenic line (KW-Bph36-NIL) in response to BPH feeding.

Brown planthopper (BPH) is the most devastating pest of rice in Asia, causing substantial yield losses and has become a challenging task to be controlled under field conditions. Although extensive measures have been taken over the past decades, which resulted in the evolution of new resistant BPH strains. Therefore, besides other possible approaches, equipping host plants with resistant genes is the most effective and environment-friendly technique for BPH control. Here, we systematically analyzed transcriptome changes in the susceptible rice variety Kangwenqingzhan (KW) and the resistant near-isogenic line (NIL) KW-Bph36-NIL, through RNA-seq, depicting the differential expression profiles of mRNAs and long non-coding RNAs (lncRNAs) in rice before and after BPH feeding. We observed a proportion of genes (1.48%) and (2.74%) were altered in KW and NIL, respectively, indicating different responses of rice strains against BPH feeding. Nevertheless, we characterized 384 differentially expressed long non-coding RNAs (DELs) that can be impacted by the two strains by alternatively changing the expression patterns of the respective coding genes, suggesting their certain involvement in response to BPH feeding. In BPH invasion, KW and NIL responded differently by modifying the synthesis, storage, and transformation of intracellular substances, adjusting the nutrient accumulation and utilization inside and outside the cells. In addition, NIL expressed stronger resistance by acutely up-regulating genes and other transcription factors related to stress resistance and plant immunity. Altogether, our study elaborates valuable insights into the genome-wide DEGs and DELs expression profiles of rice under BPH invasion by high throughput sequencing and further suggests that NILs can be utilized in BPH resistance breeding programs in developing high-resistance rice lines.

Stem and leaf rust-induced miRNAome in bread wheat near-isogenic lines and their comparative analysis.

Wheat rusts remain a major threat to global wheat production and food security. The R-gene-mediated resistance has been employed as an efficient approach to develop rust-resistant varieties. However, evolution of new fungal races and infection strategies put forward the urgency of unravelling novel molecular players, including non-coding RNAs for plant response. This study identified microRNAs associated with Sr36 and Lr45 disease resistance genes in response to stem and leaf rust, respectively. Here, small RNA sequencing was performed on susceptible and resistant wheat near-isogenic lines inoculated with stem and leaf rust pathotypes. microRNA mining in stem rust-inoculated cultivars revealed a total of distinct 26 known and 7 novel miRNAs, and leaf rust libraries culminated with 22 known and 4 novel miRNAs. The comparative analysis between two disease sets provides a better understanding of altered miRNA profiles associated with respective R-genes and infections. Temporal differential expression pattern of miRNAs pinpoints their role during the progress of infection. Differential expression pattern of miRNAs among various treatments as well as time-course expression of miRNAs revealed stem and leaf rust-responsive miRNAs and their possible role in balancing disease resistance/susceptibility. Disclosure of guide strand, passenger strand and a variant of novel-Tae-miR02 from different subgenome origins might serve as a potential link between stem and leaf rust defence mechanisms downstream to respective R-genes. The outcome from the analysis of microRNA dynamics among two rust diseases and further characterization of identified microRNAs can contribute to significant novel insights on wheat-rust interactions and rust management. KEY POINTS: • Identification and comparative analysis of stem and leaf rust-responsive miRNAs. • Chromosomal location and functional prediction of miRNAs. • Time-course expression analysis of pathogen-responsive miRNAs.

Identification and Mapping of bs8, a Novel Locus Conferring Resistance to Bacterial Spot Caused by Xanthomonas gardneri.

Although cultivars possessing recessive resistance alleles provide effective control of bacterial spot of pepper (Capsicum annuum), the deployed resistance gene, bs5, is ineffective against Xanthomonas gardneri, one of the pathogenic species. Resistance against X. gardneri was identified in C. annuum accession PI 163192, and this study sought to characterize this novel resistance and to map the resistance gene(s) to the pepper genome. We crossed PI 163192 with the susceptible cultivar Early Calwonder (ECW) to develop resistant near-isogenic lines (NILs) of ECW, designated ECW80R. The novel resistance in ECW80R was determined to be quantitative, recessively inherited, and non-hypersensitive-response causing, and inhibits lesion expansion and chlorosis. Presence of the resistance in NILs decreased the in planta bacterial population by ninefold compared with ECW. Bulked segregant analysis of resistant and susceptible individuals from an F2 population using whole genome single nucleotide polymorphisms identified a major resistance locus within an approximate 6-Mbp interval on the subtelomeric region of chromosome 11. We developed markers spanning this region and used these to genotype backcross F2 populations, which further delimited the resistance locus within a 2.3-Mbp interval. The novel resistance locus has been designated bs8. ECW80R and the linked markers developed in this study should prove useful for breeders seeking to advance this resistance into commercially relevant germplasm and for pyramiding bs8 with other resistance alleles such as bs5 and bs6. The allele bs8 will help prolong the durability of bacterial spot resistance in pepper and improve resistance to multiple species of Xanthomonas.

Multi-Omics Analysis Reveals a Regulatory Network of ZmCCT During Maize Resistance to Gibberella Stalk Rot at the Early Stage.

Gibberella stalk rot (GSR) caused by Fusarium graminearum is one of the most devastating diseases in maize; however, the regulatory mechanism of resistance to GSR remains largely unknown. We performed a comparative multi-omics analysis to reveal the early-stage resistance of maize to GSR. We inoculated F. graminearum to the roots of susceptible (Y331) and resistant (Y331-ΔTE) near-isogenic lines containing GSR-resistant gene ZmCCT for multi-omics analysis. Transcriptome detected a rapid reaction that confers resistance at 1–3 hpi as pattern-triggered immunity (PTI) response to GSR. Many key properties were involved in GSR resistance, including genes in photoperiod and hormone pathways of salicylic acid and auxin. The activation of programmed cell death-related genes and a number of metabolic pathways at 6 hpi might be important to prevent further colonization. This is consistent with an integrative analysis of transcriptomics and proteomics that resistant-mediated gene expression reprogramming exhibited a dynamic pattern from 3 to 6 hpi. Further metabolomics analysis revealed that the amount of many chemical compounds was altered in pathways associated with the phenylpropanoid biosynthesis and the phenylalanine metabolism, which may play key roles to confer the GSR resistance. Taken together, we generated a valuable resource to interpret the defense mechanism during early GSR resistance.

The effect of the Rht1 haplotype on Fusarium head blight resistance in relation to type and level of background resistance and in combination with Fhb1 and Qfhs.ifa-5A

Key messageThe effect of the Rht1-genes on FHB resistance depends on anther extrusion and level of background resistance. Qfhs.ifa-5A increases resistance and anther extrusion as efficiently as semi-dwarfing alleles decrease it.The semi-dwarfing reduced height alleles Rht-D1b and Rht-B1b have been deployed in modern wheat cultivars throughout the world, but they increase susceptibility to Fusarium head blight (FHB). Here, we investigated the impact of the Rht1 genes on anther retention (AR) in relation to FHB resistance using four different sets of near-isogenic lines (NILs) with contrasting levels and types of background FHB resistance. NILs were evaluated for FHB severity, plant height and AR in three greenhouse and three field trials using artificial spray inoculation. Rht-B1b and Rht-D1b alleles increased AR and FHB susceptibility in all genetic backgrounds. The magnitude of the effects differed between NIL groups. Increased FHB susceptibility largely followed increased AR. Differences in FHB susceptibility between tall and dwarf haplotypes were largest in the NIL group with the highest changes in AR. In the most resistant NIL group, dwarfed lines had only slightly higher AR than tall lines and maintained good resistance, while both tall and dwarf lines had high levels of retained anthers in the most susceptible NIL group. We further investigated the effect of the major Fusarium resistance QTL Fhb1 and Qfhs.ifa-5A in combination with the Rht1 genes. Qfhs.ifa-5A enhanced anther extrusion in tall as well as semi-dwarf haplotypes, whereas Fhb1 did not affect AR. Qfhs.ifa-5A supported FHB resistance more efficiently than Fhb1 in lines that were more responsive to AR, while both Fhb1 and Qfhs.ifa-5A were equally efficient in NILs that had high background resistance and low response to AR.

Analysis of immune response-related LRR-RLKs differential genes in the rice blast resistance near-isogenic lines of <italic>Japonica</italic> Variety Yunyin

Rice ( Oryza sativa L.) is a staple food in the world as well as an important monocotyledonous model plant for molecular biology, which has a long history of cultivation and domestication. However, many diseases will break out during rice production. For example, the rice blast is the most destructive disease, which can cause the heavy loss or even total crop failure. Therefore, there are constant demands to cultivate durably blast-resistant varieties and identify more broad-spectrum blast resistance genes. Besides, it’s also very significant for agricultural production to elucidate the process and mechanism of immune response during the rice blast infection. The whole-genome sequencing of the resistant and susceptible near-isogenic lines derived from the broad-spectrum blast resistance Japonica Variety Yunyin and the universally blast-susceptible variety Lijiangxintuanheigu (LTH) was completed. Eight differential genes were screened from the Go terms that were associated with the immune response based on the whole-genome sequence data. We analyzed their gene structure, evolution and sequence variability between the resistant and susceptible near-isogenic lines. The results indicated that the homology of these protein sequences was between 55% and 80%, and the conserved protein domain analysis showed that most of them contained different numbers and types of leucine-rich repeat domains, a transmembrane domain and a C-terminal kinase domain, which were typical leucine-rich repeat receptor-like protein kinases. Moreover, they are all located within a cluster of chromosome 11 and were homologous to the bacterial blight resistance gene Xa21 . The leucine-rich repeat receptor-like protein kinases (LRR-RLKs) genes are a large conserved superfamily during the plant evolution. In Arabidopsis , there are 14 subfamilies and more than 200 members of LRR-RLKs were identified, whereas the rice genome contained more than 900 LRR-RLKs genes. The perception and recognition of pathogen infection are mainly mediated by the plant cells surface pattern-recognition receptors (PRRs) and intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors. PRRs contain many receptor kinases including the leucine-rich repeat receptor-like protein kinases, which play an important role in the signal transduction during the plant development, immune response and sense of the environmental changes. In order to further explore the potential function of these genes during the rice blast disease response, the rice seedlings were inoculated with Magnaporthe oryzae by spray and punch methods and then the expression pattern of these genes during the fungal infection were analyzed by qRT-PCR. The results showed that some genes response to the blast disease. Thus, we speculated that some of them might be involved in the rice blast disease response. This study laid a foundation for further studying genes related to the rice blast disease response.

A study of miRNAs and lncRNAs during Lr28-mediated resistance against leaf rust in wheat (Triticum aestivum L.)

Wheat (Triticum aestivum L.) is a major crop, which is widely grown, occupying globally an area of ~225 million hectares. The crop suffers major losses due to leaf rust caused by fungal pathogen, Puccinia triticina Eriks. and E. Henn. When wheat plants are attacked by this fungus, hundreds of downstream genes involved in signal transduction pathways experience regulated expression. This regulation partly depends on non-coding RNAs (ncRNAs) including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). In the present study, miRNAs and lncRNAs associated with wheat-leaf rust pathosystem were identified using RNA-seq data for a pair of near-isogenic lines (NILs), which differed for the gene Lr28 in the background of wheat cultivar HD2329. The study is a continuation of our earlier transcriptome study involving the same pathosystem. A total of 50 miRNAs and 1178 lncRNAs were identified through in silico analysis of RNA-seq data; of these, 16 miRNAs and 22 lncRNAs were differentially expressed (DE). Expression of as many as 8 miRNAs was induced in resistant NIL; these DE miRNAs targeted several important genes, which include disease responsive genes. As many as 49 lncRNAs were found to be the targets for miRNAs; among these, one lncRNA functioned as a precursor of two mature miRNAs, and three lncRNAs acted as target mimics (TMs, which mimic and therefore compete with the mRNA targets for miRNA) thus regulating the expression of target genes. The results were also validated using qRT-PCR analyses. Taken together, the leaf rust responsive miRNAs, their targets along with putative TMs, and the lncRNAs, identified in the present study, should improve our understanding about the role of non-coding RNAs during response to leaf rust in wheat.

Deciphering signalling network in broad spectrum Near Isogenic Lines of rice resistant to Magnaporthe oryzae

Disease resistance (R) genes like Pi9, Pita, Pi21, Pi54 are playing important role for broad spectrum blast resistance in rice. Development of near isogenic lines (NILs) using these type of broad spectrum genes and understanding their signalling networks is essential to cope up with highly evolving Magnaporthe oryzae strains for longer duration. Here, transcriptional-level changes were studied in three near-isogenic lines (PB1 + Pi1, PB1 + Pi9 and PB1 + Pi54) of rice resistant to blast infection, to find the loci that are unique to resistant lines developed in the background of Pusa Basmati 1 (PB1). The pathway analysis of loci, unique to resistant NILs compared to susceptible control revealed that plant secondary metabolite synthesis was the common mechanism among all NILs to counter against M. oryzae infection. Comparative transcriptome analysis helped to find out common clusters of co-expressed significant differentially expressed loci (SDEL) in both PB1 + Pi9 and PB1 + Pi54 NILs. SDELs from these clusters were involved in the synthesis and degradation of starch; synthesis and elongation of fatty acids; hydrolysis of phospholipids; synthesis of phenylpropanoid; and metabolism of ethylene and jasmonic acid. Through detailed analysis of loci specific to each resistant NIL, we identified a network of signalling pathways mediated by each blast resistance gene. The study also offers insights into transcriptomic dynamics, points to a set of important candidate genes that serve as module to regulate the changes in resistant NILs. We suggest that pyramiding of the blast resistance gene Pi9 with Pi54 will lead to maximum broad spectrum resistance to M. oryzae.

Candidate genes expression profiling during wilting in chickpea caused by Fusarium oxysporum f. sp. ciceris race 5.

Chickpea production may be seriously threatened by Fusarium wilt, a disease caused by the soil-borne fungus Fusarium oxysporum f. sp. ciceris. F. oxysporum race 5 is the most important race in the Mediterranean basin. Recently, the region responsible for resistance race 5 has been delimited within a region on chromosome 2 that spans 820 kb. To gain a better understanding of this genomic region, we used a transcriptomic approach based on quantitative real-time PCR to analyze the expression profiles of 22 selected candidate genes. We used a pair of near-isogenic lines (NILs) differing in their sensitivity to Fusarium race 5 (resistant vs susceptible) to monitor the transcriptional changes over a time-course experiment (24, 48, and 72 hours post inoculation, hpi). Qualitative differences occurred during the timing of regulation. A cluster of 12 genes were induced by the resistant NIL at 24 hpi, whereas a second cluster contained 9 genes induced by the susceptible NIL at 48 hpi. Their possible functions in the molecular defence of chickpea is discussed. Our study provides new insight into the molecular defence against Fusarium race 5 and demonstrates that development of NILs is a rich resource to facilitate the detection of candidate genes. The new genes regulated here may be useful against other Fusarium races.

Microbiome responses during virulence adaptation by a phloem-feeding insect to resistant near-isogenic rice lines.

The microbiomes of phloem‐feeding insects include functional bacteria and yeasts essential for herbivore survival and development. Changes in microbiome composition are implicated in virulence adaptation by herbivores to host plant species or host populations (including crop varieties). We examined patterns in adaptation by the green leafhopper, Nephotettix virescens, to near‐isogenic rice lines (NILs) with one or two resistance genes and the recurrent parent T65, without resistance genes. Only the line with two resistance genes was effective in reducing leafhopper fitness. After 20 generations on the resistant line, selected leafhoppers attained similar survival, weight gain, and egg laying to leafhoppers that were continually reared on the susceptible recurrent parent, indicating that they had adapted to the resistant host. By sequencing the 16s rRNA gene, we described the microbiome of leafhoppers from colonies associated with five collection sites, and continually reared or switched between NILs. The microbiomes included 69–119 OTUs of which 44 occurred in ≥90% of samples. Of these, 14 OTUs were assigned to the obligate symbiont Candidatus sulcia clade. After 20 generations of selection, collection site had a greater effect than host plant on microbiome composition. Six bacteria genera, including C. sulcia, were associated with leafhopper virulence. However, there was significant within‐treatment, site‐related variability in the prevalence of these taxa such that the mechanisms underlying their association with virulence remain to be determined. Our results imply that these taxa are associated with leafhopper nutrition. Ours is the first study to describe microbiome diversity and composition in rice leafhoppers. We discuss our results in light of the multiple functions of herbivore microbiomes during virulence adaptation in insect herbivores.

Fine-mapping of the Fusarium head blight resistance QTL Qfhs.ifa-5A identifies two resistance QTL associated with anther extrusion

Key messageFine-mapping separated Qfhs.ifa-5A into a major QTL mapping across the centromere and a minor effect QTL positioned at the distal half of 5AS. Both increase Fusarium resistance and anther extrusion.The Fusarium head blight (FHB) resistance QTL Qfhs.ifa-5A resides in the low-recombinogenic pericentromeric region of chromosome 5A making fine-mapping particularly arduous. Qfhs.ifa-5A primarily contributes resistance to fungal entry with the favorable allele descending from the highly Fusarium resistant cultivar Sumai-3. Fine-mapping a near-isogenic recombinant inbred line population partitioned the Qfhs.ifa-5A interval into 12 bins. Near-isogenic lines recombining at the interval were phenotyped for FHB severity, anther retention and plant height. Composite interval mapping separated the initially single QTL into two QTL. The major effect QTL Qfhs.ifa-5Ac mapped across the centromere and the smaller effect QTL Qfhs.ifa-5AS mapped to the distal half of 5AS. Although Qfhs.ifa-5Ac and Qfhs.ifa-5AS intervals were as small as 0.1 and 0.2 cM, their corresponding physical distances were large, comprising 44.1 Mbp and 49.2 Mbp, respectively. Sumai-3 alleles at either QTL improved FHB resistance and increased anther extrusion suggesting a pleiotropic effect of anthers on resistance. This hypothesis was supported by greenhouse experiments using the susceptible cultivar Remus and its resistant near-isogenic line NIL3 carrying the entire Qfhs.ifa-5A segment. By manually removing anthers prior to spray inoculation both, Remus and NIL3 became almost equally resistant in the early phase of the disease development and were significantly less diseased than variants without anther manipulation. At late time points the positive effect of the anther removal became smaller for Remus and disappeared completely for NIL3. Results affirm that absence of anthers enhanced resistance to initial infection but did not protect plants from fungal spreading within spikes.

Cytochrome P450 monooxygenases-mediated sex-differential spinosad resistance in house flies Musca domestica (Diptera: Muscidae)

Females developed notably higher resistance than males in a spinosad-resistant house fly strain, however, resistance factors responsible for this phenomenon are poorly understood. In this study, the potential role of cytochrome P450 monooxygenases involved in the sex-differential spinosad resistance in house flies was investigated, using a susceptible strain (SSS) and a spinosad resistant near-isogenic line (N-SRS). Combination of the synergism of spinosad by PBO and increased cytochrome P450 monooxygenase activity in the N-SRS strain implied that cytochrome P450 monooxygenases contributed to spinosad resistance in house flies. Transcriptional levels of eight P450 genes related to insecticide resistance in two genders of the SSS and N-SRS strain were separately evaluated by quantitative real-time PCR. Notably, compared with the corresponding gender of susceptible SSS house flies, CYP4G2 and CYP6A5v2 were overexpressed in resistant N-SRS females, while the expression of these two P450 genes was significantly decreased in resistant N-SRS males. Furthermore, by measuring the expression of CYP4G2 and CYP6A5v2 in female and male house fly populations with different spinosad resistance levels, which were generated from a series of genetic crosses, the genetic linkage between spinosad resistance and P450 gene expression was analyzed. It was found that with increased spinosad resistance, CYP4G2 and CYP6A5v2 were up-regulated in females, while both of them were down-regulated in males, and this suggested their involvement in the female-linked spinosad resistance of house flies. Taken together, our results provide valuable insight into the involvement of cytochrome P450 monooxygenases in the sex-differential spinosad resistance in house flies.

Feature of blast resistant near-isogenic lines using an elite maintainer line II-32B by marker-assisted selection

Rice plays a crucial role in food production worldwide as one of the most important cereal crops. Breeding blast disease resistant varieties is a long-term aim for sustainable rice production. II-32B is an elite maintainer line of II-32A which has been widely used as a hybrid rice parent in China. However, it is significantly susceptible to rice blast. In this study, multiple alleles, Pi-2 and Pi-9, and the chromosomal segment of blast resistance genes, Pi-1 and Pi-kh, were introgressed to receptor parents II-32B by marker-assisted backcross breeding. Eleven near-isogenic lines (NILs) with single Pi-gene and 12 pyramid lines containing double or three homozygous resistance genes were obtained. These improved lines were identified by inoculating Magnaporthe oryzae isolates in the field/greenhouses; the result showed that the resistance of pyramid lines containing three resistance genes was stronger than the lines with double or single gene, whereas the lines with double genes were stronger than the one with single-gene. Additionally, pyramiding of different Pi-genes was effective in obtaining the durable blast resistance in rice. This implies that gene pyramiding in rice can increase spectrum and strength of resistance to blast. Furthermore, three-line retention of sixteen improved NILs was kept well and the agronomic traits were consistence with II-32B. We conclude that gene pyramiding is an effective breeding approach for the improvement of rice varieties with durable resistance to blast in the future.

Resistant silkworm strain block viral infection independent of melanization

Melanization mediated by the prophenoloxidase-activating system (proPO) is an important immune response in invertebrates. However, the role of melanization on viral infection has not been wildly revealed in Bombyx mori (B. mori), silkworm. Here, we investigated the extent of melanization of susceptible (871) and resistant (near-isogenic line 871C) B. mori strains. The result showed that the extent of melanization was significantly higher in the susceptible strain than in the resistant strain. A majority of Serpins were up-regulated in the resistant strain through iTRAQ-based quantitative proteomics, comparing with susceptible strain. Our data further identified that Serpin-5, Serpin-9 and Serpin-19 reduced PO activity, indicating that the menlanization pathway was inhibited in the resistant strain. Moreover, our results indicated that the hemolymph of 871 reduced viral infection in the presence of PTU, indicating that melanization of 871 strain hemolymph blocked viral infection. However, viral infection was significantly suppressed in the hemolymph of 871C strain regardless of the presence of PTU or not, which implied that the resistant strain inhibited viral infection independent of the melanization pathway. Taken together, our findings indicated that the melanization pathway was inhibited in resistant strain. These results expend the analysis of melanization pathway in insects and provide insights into understanding the antiviral mechanism.

Stage-specific reprogramming of defense responsive genes during Lr24-mediated leaf rust resistance in wheat

Resistance expressed by plants against different rust pathogens is a unique and a very well-coordinated process. The defense responsive genes are the performers of such responses in plants. Understanding the regulation and mechanism of action of these genes is a key constituent of strategic resistance breeding programmes. The present study is aimed at evaluating and comparing the temporal expression pattern of defense responsive genes during early infection phase in compatible and incompatible interactions between wheat and leaf rust to elucidate the molecular basis of Lr24- mediated rust resistance. Lr24 confers resistance to almost all the pathotypes of Puccinia triticina (Pt) reported from Indian subcontinent and Australia. Expression profiles of 10 pathogen responsive genes representing different classes were studied in susceptible (HS240) and resistant (HW2020) wheat near-isogenic lines interacting with Pt. The expression profiles of these genes revealed that the transcript levels of aquaporin, endochitinase, β-1,3-glucanase, Type 1 non-specific lipid transfer protein precursor, phenylalanine ammonia-lyase and caffeic acid O-methyltransferase were significantly higher in incompatible interaction. Relatively higher expression of phenylalanine ammonia-lyase and aquaporin was observed at the pre-haustorial stage [6 h post inoculation (hpi) and 12 hpi], while expression of endo-chitinase and lipid transfer protein was higher during the post-haustorial stage (24 hpi and 48 hpi) in the incompatible interaction. Induced expression of class III peroxidase was maintained throughout pre- and post-haustorial stages in the compatible interaction. The results of the current study could distinguish the defense responsive genes playing a critical role in wheat resistance or susceptibility to leaf rust. We feel that this understanding will allow devising novel strategies to regulate wheat leaf rust pathogen interactions through genetic engineering.

Effect of Ahasl1-1 and Ahasl1-4 alleles on herbicide resistance and its associated dominance in sunflower.

Acetohydroxyacid synthase large subunit 1 (Ahasl1) is a multiallelic locus involved in herbicide resistance in sunflower. Ahasl1-1 and Ahasl1-4 alleles harbor different point mutations that lead to different amino acid substitutions (Ala205Val and Trp574Leu, respectively). The objectives of this work were to evaluate the effect of these alleles at the enzymatic and whole-plant levels, and to determine the dominance relationships for imazapyr and metsulfuron-methyl herbicides. Resistant near-isogenic lines showed significantly lower specific AHAS activity than susceptible near-isoline. However, kinetic studies indicated that mutations did not change AHAS pyruvate affinity. Dose-response for six near-isolines carrying different combinations of Ahasl1-1 and Ahasl1-4 alleles and two herbicides (imazapyr and metsulfuron-methyl) were evaluated at whole-plant and enzymatic levels. Ahasl1-1 allele conferred moderate resistance to imazapyr and low resistance to metsulfuron-methyl. Conversely, Ahasl1-4 allele endowed high levels of resistance for both herbicides. Dominance of resistance at whole-plant level showed a semi-dominant behavior among the alleles for both herbicides. Ahasl1-4 allele confers higher resistance levels than Ahasl1-1 when evaluated with imazapyr and metsulfuron-methyl. Dominance estimations suggested that both parental lines should carry a resistance trait when developing hybrids. © 2018 Society of Chemical Industry.

H3K4/K9 acetylation and Lr28-mediated expression of six leaf rust responsive genes in wheat (Triticum aestivum).

Development of leaf rust-resistant cultivars is a priority during wheat breeding, since leaf rust causes major losses in yield. Resistance against leaf rust due to Lr genes is partly controlled by epigenetic modifications including histone acetylation that is known to respond to biotic/abiotic stresses. In the present study, enrichment of H3K4ac and H3K9ac in promoters of six defense responsive genes (N-acetyltransferase, WRKY 40, WRKY 70, ASR1, Peroxidase 12 and Sarcosine oxidase) was compared with their expression in a pair of near-isogenic lines (NILs) for the gene Lr28 following inoculation with leaf rust pathotype '77-5'; ChIP-qPCR was used for this purpose. The proximal and distal promoters of these genes contained a number of motifs that are known to respond to biotic stresses. The enrichment of two acetylation marks changed with passage of time; changes in expression of two of the six genes (N-acetyltransferase and peroxidase12), largely matched with changes in H3K4/H3K9 acetylation patterns of the two promoter regions. For example, enrichment of both the marks matched with higher expression of N-acetyltransferase gene in susceptible NIL and the deacetylation (H3K4ac) largely matched with reduced gene expression in resistant NIL. In peroxidase12, enrichment of H3K4ac and H3K9ac largely matched with higher expression in both the NILs. In the remaining four genes, changes in H3 acetylation did not always match with gene expression levels. This indicated complexity in the regulation of the expression of these remaining four genes, which may be controlled by other epigenetic/genetic regulatory mechanisms that need further analysis.