Functional analysis revealed the involvement of ZmABCB15 in resistance to rice black-streaked dwarf virus infection.

BACKGROUND: Maize rough dwarf disease (MRDD), caused by rice black-streaked dwarf virus (RBSDV) belonging to the Fijivirus genus, seriously threatens maize production worldwide. Three susceptible varieties (Ye478, Zheng 58, and Zhengdan 958) and two resistant varieties (P138 and Chang7-2) were used in our study. RESULTS: A set of ATP-binding cassette subfamily B (ABCB) transporter genes were screened to evaluate their possible involvements in RBSDV resistance. In the present study, ZmABCB15, an ABCB transporter family member, was cloned and functionally identified. Expression analysis showed that ZmABCB15 was significantly induced in the resistant varieties, not in the susceptible varieties, suggesting its involvement in resistance to the RBSDV infection. ZmABCB15 gene encodes a putative polar auxin transporter containing two trans-membrane domains and two P-loop nucleotide-binding domains. Transient expression analysis indicated that ZmABCB15 is a cell membrance localized protein. Over-expression of ZmABCB15 enhanced the resistance by repressing the RBSDV replication ratio. ZmABCB15 might participate in the RBSDV resistance by affecting the homeostasis of active and inactive auxins in RBSDV infected seedlings. CONCLUSIONS: Polar auxin transport might participate in the RBSDV resistance by affecting the distribution of endogenous auxin among tissues. Our data showed the involvement of polar auxin transport in RBSDV resistance and provided novel mechanism underlying the auxin-mediated disease control technology.

Identification and Fine-Mapping of a Novel QTL, qMrdd2, That Confers Resistance to Maize Rough Dwarf Disease.

Maize rough dwarf disease (MRDD) is caused by a virus and seriously affects maize quality and yield worldwide. MRDD can be most effectively controlled with disease-resistant hybrids of corn. Here, MRDD-resistant (Qi319) and -susceptible (Ye478) parental inbred maize lines and their 314 recombinant inbred lines (RILs) that were derived from a cross between them were evaluated across three environments. A stable resistance QTL, qMrdd2, was identified and mapped using best linear unbiased prediction (BLUP) values to a 0.55-Mb region between the markers MK807 and MK811 on chromosome 2 (B73 RefGen_v3) and was found to explain 8.6 to 11.0% of the total phenotypic variance in MRDD resistance. We validated the effect of qMrdd2 using a chromosome segment substitution line (CSSL) that was derived from a cross between maize inbred Qi319 as the MRDD resistance donor and Ye478 as the recipient. Disease severity index of the CSSL haplotype II harboring qMrdd2 was significantly lower than that of the susceptible parent Ye478. Subsequently, we fine-mapped qMrdd2 to a 315-kb region flanked by the markers RD81 and RD87, thus testing recombinant-derived progeny using selfed backcrossed families. In this study, we identified a novel QTL for MRDD resistance by combining the RIL and CSSL populations, thus providing important genetic information that can be used for breeding MRDD-resistant varieties of maize.

Marker-assisted selection of qMrdd8 to improve maize resistance to rough dwarf disease.

Maize rough dwarf disease (MRDD) is caused by viruses in the Fijivirus genus in the family Reoviridae. MRDD resistance can be improved by a combination of marker-assisted selection (MAS) and conventional breeding strategies. In our previous study, we fine-mapped a major QTL qMrdd8 and developed the functional Indel marker IDP25K. In the present study, qMrdd8 from the donor parent X178 was introgressed into elite inbred lines derived from the three corn heterotic groups using multi-generation backcrossing and MAS. Recipient lines included Huangzao4, Chang7-2, Ye478, Zheng58, Zhonghuang68, B73, and Ji846. Markers used for foreground selection included IDRQ4, IDRQ47, IDP25K, and IDP27K. Background selection was carried out in the BC3 or BC4 using 107 SSR markers to select lines with the highest rate of recovery of the particular recurrent parent genome. Plants from BC4F2 and BC3F2 that carried the shortest qMrdd8 interval from X178 and those with the highest rate of recovery of the recurrent parent genome were then selected to create converted homozygous inbred lines. In 2017, seven converted inbred lines and five hybrids exhibited enhanced resistance to MRDD, while other agronomic traits were not affected under nonpathogenic stress conditions. Thus, the MRDD resistance allele at the qMrdd8 locus, or IDP25K, should be valuable for maize breeding programs in China.

Comparative proteomic analysis of maize (Zea mays L.) seedlings under rice black-streaked dwarf virus infection.

BACKGROUND: Maize rough dwarf disease (MRDD) is a severe disease that has been occurring frequently in southern China and many other Asian countries. MRDD is caused by the infection of Rice black streaked dwarf virus (RBSDV) and leads to significant economic losses in maize production. To well understand the destructive effects of RBSDV infection on maize growth, comparative proteomic analyses of maize seedlings under RBSDV infection was performed using an integrated approach involving LC-MS/MS and Tandem Mass Tag (TMT) labeling. RESULTS: In total, 7615 maize proteins, 6319 of which were quantified. A total of 116 differentially accumulated proteins (DAPs) were identified, including 35 up- and 81 down-regulated proteins under the RBSDV infection. Enrichment analysis showed that the DAPs were most strongly associated with cyanoamino acid metabolism, protein processing in ER, and ribosome-related pathways. Two sulfur metabolism-related proteins were significantly reduced, indicating that sulfur may participate in the resistance against RBSDV infection. Furthermore, 15 DAPs involved in six metabolic pathways were identified in maize under the RBSDV infection. CONCLUSIONS: Our data revealed that the responses of maize to RBSDV infection were controlled by various metabolic pathways.

Identification and Analysis of Medicago truncatula Auxin Transporter Gene Families Uncover their Roles in Responses to Sinorhizobium meliloti Infection.

Auxin transport plays a pivotal role in the interaction between legume species and nitrogen-fixing bacteria to form symbioses. Auxin influx carriers auxin resistant 1/like aux 1 (AUX/LAX), efflux carriers pin-formed (PIN) and efflux/conditional P-glycoprotein (PGP/ABCB) are three major protein families participating in auxin polar transport. We used the latest Medicago truncatula genome sequence to characterize and analyze the M. truncatula LAX (MtLAX), M. truncatula PIN (MtPIN) and M. truncatula ABCB (MtABCB) families. Transient expression experiments indicated that three representative auxin transporters (MtLAX3, MtPIN7 and MtABCB1) showed cell plasma membrane localizations. The expression of most MtLAX, MtPIN and MtABCB genes was up-regulated in the roots and was down-regulated in the shoots by Sinorhizobium meliloti infection in the wild type (WT). However, the expression of these genes was down-regulated in both the roots and shoots of an infection-resistant mutant, dmi3. The different expression patterns between the WT and the mutant roots indicated that auxin relocation may be involved in rhizobial infection responses. Furthermore, IAA contents were significantly up-regulated in the shoots and down-regulated in the roots after Sinorhizobium meliloti infection in the WT. Inoculation of roots with rhizobia may reduce the auxin loading from shoots to roots by inhibiting the expression of most auxin transporter genes. However, the rate of change of gene expression and IAA contents in the dmi3 mutant were obviously lower than in the WT. The identification and expression analysis of auxin transporter genes helps us to understand the roles of auxin in the regulation of nodule formation in M. truncatula.

A transcription factor ZmGLK36 confers broad resistance to maize rough dwarf disease in cereal crops.

Maize rough dwarf disease (MRDD), caused by maize rough dwarf virus (MRDV) or rice black-streaked dwarf virus (RBSDV), seriously threatens worldwide production of all major cereal crops, including maize, rice, wheat and barley. Here we report fine mapping and cloning of a previously reported major quantitative trait locus (QTL) (qMrdd2) for RBSDV resistance in maize. Subsequently, we show that qMrdd2 encodes a G2-like transcription factor named ZmGLK36 that promotes resistance to RBSDV by enhancing jasmonic acid (JA) biosynthesis and JA-mediated defence response. We identify a 26-bp indel located in the 5' UTR of ZmGLK36 that contributes to differential expression and resistance to RBSDV in maize inbred lines. Moreover, we show that ZmDBF2, an AP2/EREBP family transcription factor, directly binds to the 26-bp indel and represses ZmGLK36 expression. We further demonstrate that ZmGLK36 plays a conserved role in conferring resistance to RBSDV in rice and wheat using transgenic or marker-assisted breeding approaches. Our results provide insights into the molecular mechanisms of RBSDV resistance and effective strategies to breed RBSDV-resistant cereal crops.

Transcriptome Analysis of Cadmium-Treated Roots in Maize (Zea mays L.).

Cadmium (Cd) is a heavy metal and is highly toxic to all plant species. However, the underlying molecular mechanism controlling the effects of auxin on the Cd stress response in maize is largely unknown. In this study, the transcriptome produced by maize 'Zheng 58' root responses to Cd stress was sequenced using Illumina sequencing technology. In our study, six RNA-seq libraries yielded a total of 244 million clean short reads and 30.37 Gb of sequence data. A total of 6342 differentially expressed genes (DEGs) were grouped into 908 Gene Ontology (GO) categories and 198 Kyoto Encyclopedia of Genes and Genomes terms. GO term enrichment analysis indicated that various auxin signaling pathway-related GO terms were significantly enriched in DEGs. Comparison of the transcript abundances for auxin biosynthesis, transport, and downstream response genes revealed a universal expression response under Cd treatment. Furthermore, our data showed that free indole-3-acetic acid (IAA) levels were significantly reduced; but IAA oxidase activity was up-regulated after Cd treatment in maize roots. The analysis of Cd activity in maize roots under different Cd and auxin conditions confirmed that auxin affected Cd accumulation in maize seedlings. These results will improve our understanding of the complex molecular mechanisms underlying the response to Cd stress in maize roots.

Identification and expression profiling analysis of calmodulin-binding transcription activator genes in maize (Zea mays L.) under abiotic and biotic stresses.

The calmodulin-binding transcription activators (CAMTA) play critical roles in plant growth and responses to environmental stimuli. However, how CAMTAs function in responses to abiotic and biotic stresses in maize (Zea mays L.) is largely unknown. In this study, we first identified all the CAMTA homologous genes in the whole genome of maize. The results showed that nine ZmCAMTA genes showed highly diversified gene structures and tissue-specific expression patterns. Many ZmCAMTA genes displayed high expression levels in the roots. We then surveyed the distribution of stress-related cis-regulatory elements in the -1.5 kb promoter regions of ZmCAMTA genes. Notably, a large number of stress-related elements present in the promoter regions of some ZmCAMTA genes, indicating a genetic basis of stress expression regulation of these genes. Quantitative real-time PCR was used to test the expression of ZmCAMTA genes under several abiotic stresses (drought, salt, and cold), various stress-related hormones [abscisic acid, auxin, salicylic acid (SA), and jasmonic acid] and biotic stress [rice black-streaked dwarf virus (RBSDV) infection]. Furthermore, the expression pattern of ZmCAMTA genes under RBSDV infection was analyzed to investigate their potential roles in responses of different maize cultivated varieties to RBSDV. The expression of most ZmCAMTA genes responded to both abiotic and biotic stresses. The data will help us to understand the roles of CAMTA-mediated Ca(2+) signaling in maize tolerance to environmental stresses.

Genome-wide identification and expression analysis of auxin response factor gene family in Medicago truncatula.

Auxin response factors (ARFs) bind specifically to auxin response elements (AuxREs) in the promoters of down-stream target genes and play roles in plant responses to diverse environmental factors. Using the latest updated Medicago truncatula reference genome sequence, a comprehensive characterization and analysis of 24 MtARF (M. truncatula ARF) genes were performed. To uncover the basic information and functions of MtARF genes during symbiosis, we analyzed the expression patterns of MtARF genes during the early phase of Sinorhizobium meliloti infection. The systematic analysis indicated that changes in MtARF gene expression occur during these early stages of infection, suggesting a functional role in symbiosis. Furthermore, the roles of MtARF-mediated auxin signaling in symbiosis were tested in the infection resistant mutant (dmi3). The expression responses of MtARFs to S. meliloti infection were attenuated in the mutant compared to wild-type A17. In summary, our results show that changes in MtARF gene expression occur during the response to S. meliloti infection, suggesting that members of this family may have important roles in the symbiotic interaction.

Genome-wide identification and expression profiling analysis of ZmPIN, ZmPILS, ZmLAX and ZmABCB auxin transporter gene families in maize (Zea mays L.) under various abiotic stresses.

The auxin influx carriers auxin resistant 1/like aux 1 (AUX/LAX), efflux carriers pin-formed (PIN) (together with PIN-like proteins) and efflux/conditional P-glycoprotein (ABCB) are major protein families involved in auxin polar transport. However, how they function in responses to exogenous auxin and abiotic stresses in maize is largely unknown. In this work, the latest updated maize (Zea mays L.) reference genome sequence was used to characterize and analyze the ZmLAX, ZmPIN, ZmPILS and ZmABCB family genes from maize. The results showed that five ZmLAXs, fifteen ZmPINs, nine ZmPILSs and thirty-five ZmABCBs were mapped on all ten maize chromosomes. Highly diversified gene structures, nonconservative transmembrane helices and tissue-specific expression patterns suggested the possibility of function diversification for these genes. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the expression patterns of ZmLAX, ZmPIN, ZmPILS and ZmABCB genes under exogenous auxin and different environmental stresses. The expression levels of most ZmPIN, ZmPILS, ZmLAX and ZmABCB genes were induced in shoots and were reduced in roots by various abiotic stresses (drought, salt and cold stresses). The opposite expression response patterns indicated the dynamic auxin transport between shoots and roots under abiotic stresses. Analysis of the expression patterns of ZmPIN, ZmPILS, ZmLAX and ZmABCB genes under drought, salt and cold treatment may help us to understand the possible roles of maize auxin transporter genes in responses and tolerance to environmental stresses.

OsARF16 is involved in cytokinin-mediated inhibition of phosphate transport and phosphate signaling in rice (Oryza sativa L.).

BACKGROUND: Plant responses to phytohormone stimuli are the most important biological features for plants to survive in a complex environment. Cytokinin regulates growth and nutrient homeostasis, such as the phosphate (Pi) starvation response and Pi uptake in plants. However, the mechanisms underlying how cytokinin participates in Pi uptake and Pi signaling are largely unknown. In this study, we found that OsARF16 is required for the cytokinin response and is involved in the negative regulation of Pi uptake and Pi signaling by cytokinin. PRINCIPAL FINDINGS: The mutant osarf16 showed an obvious resistance to exogenous cytokinin treatment and the expression level of the OsARF16 gene was considerably up-regulated by cytokinin. Cytokinin (6-BA) application suppressed Pi uptake and the Pi starvation response in wild-type Nipponbare (NIP) and all these responses were compromised in the osarf16 mutant. Our data showed that cytokinin inhibits the transport of Pi from the roots to the shoots and that OsARF16 is involved in this process. The Pi content in the osarf16 mutant was much higher than in NIP under 6-BA treatment. The expressions of PHOSPHATE TRANSPORTER1 (PHT1) genes, phosphate (Pi) starvation-induced (PSI) genes and purple PAPase genes were higher in the osarf16 mutant than in NIP under cytokinin treatment. CONCLUSION: Our results revealed a new biological function for OsARF16 in the cytokinin-mediated inhibition of Pi uptake and Pi signaling in rice.

Genome-wide identification and expression profiling analysis of the Aux/IAA gene family in Medicago truncatula during the early phase of Sinorhizobium meliloti infection.

BACKGROUND: Auxin/indoleacetic acid (Aux/IAA) genes, coding a family of short-lived nuclear proteins, play key roles in wide variety of plant developmental processes, including root system regulation and responses to environmental stimulus. However, how they function in auxin signaling pathway and symbiosis with rhizobial in Medicago truncatula are largely unknown. The present study aims at gaining deeper insight on distinctive expression and function features of Aux/IAA family genes in Medicago truncatula during nodule formation. PRINCIPAL FINDINGS: Using the latest updated draft of the full Medicago truncatula genome, a comprehensive identification and analysis of IAA genes were performed. The data indicated that MtIAA family genes are distributed in all the M. truncatula chromosomes except chromosome 6. Most of MtIAA genes are responsive to exogenous auxin and express in tissues-specific manner. To understand the biological functions of MtIAA genes involved in nodule formation, quantitative real-time polymerase chain reaction (qRT-PCR) was used to test the expression profiling of MtIAA genes during the early phase of Sinorhizobium meliloti (S. meliloti) infection. The expression patterns of most MtIAA genes were down-regulated in roots and up-regulated in shoots by S. meliloti infection. The differences in expression responses between roots and shoots caused by S. meliloti infection were alleviated by 1-NOA application. CONCLUSION: The genome-wide identification, evolution and expression pattern analysis of MtIAA genes were performed in this study. The data helps us to understand the roles of MtIAA-mediated auxin signaling in nodule formation during the early phase of S. meliloti infection.