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Rice black-streaked dwarf virus is transmitted by small brown planthoppers, which causes maize rough dwarf disease and rice black-streaked dwarf disease. This virus leads to slow growth or death of the host plants. During the coevolutionary arms race between viruses and plants, virus-derived small interfering RNAs (vsiRNAs) challenge the plant's defense response and inhibit host immunity through the RNA silencing system. However, it is currently unknown if rice black-streaked dwarf virus can produce the same siRNAs to mediate the RNA silencing in different infected species. In this study, four small RNA libraries and four degradome libraries were constructed by extracting total RNAs from the leaves of the maize ( Zea mays ) inbred line B73 and japonica rice ( Oryza sativa ) variety Nipponbare exposed to feeding by viruliferous and nonviruliferous small brown planthoppers. We analyzed the characteristics of small RNAs and explored virus-derived siRNAs in small RNA libraries through high-throughput sequencing. On analyzing the characteristics of small RNA, we noted that the size distributions of small RNAs were mainly 24 nt (19.74 to 62.00%), whereas those of vsiRNAs were mostly 21 nt (41.06 to 41.87%) and 22 nt (39.72 to 42.26%). The 5'-terminal nucleotides of vsiRNAs tended to be adenine or uracil. Exploring the distribution of vsiRNA hot spots on the viral genome segments revealed that the frequency of hotspots in B73 was higher than those in Nipponbare. Meanwhile, hotspots in the S9 and S10 virus genome segments were distributed similarly in both hosts. In addition, the target genes of small RNA were explored by degradome sequencing. Analyses of the regulatory pathway of these target genes unveiled that viral infection affected the ribosome-related target genes in maize and the target genes in the metabolism and biosynthesis pathways in rice. Here, 562 and 703 vsiRNAs were separately obtained in maize and rice and 73 vsiRNAs named as coexisting vsiRNAs (co-vsiRNAs) were detected in both hosts. Stem-loop PCR and real-time quantitative PCR confirmed that co-vsiRNA 3.1 and co-vsiRNA 3.5, derived from genome segment S3, simultaneously play a role in maize and rice and inhibited host gene expression. The study revealed that rice black-streaked dwarf virus can produce the same siRNAs in different species and provides a new direction for developing new antiviral strategies., Competing Interests: The author(s) declare no conflict of interest.

Pathogen infection induces massive reprogramming of host primary metabolism. Lipid and fatty acid (FA) metabolism is generally disrupted by pathogens and co-opted for their proliferation. Lipid droplets (LDs) that play important roles in regulating cellular lipid metabolism are utilized by a variety of pathogens in mammalian cells. However, the function of LDs during pathogenic infection in plants remains unknown. We show here that infection by rice black streaked dwarf virus (RBSDV) affects the lipid metabolism of maize, which causes elevated accumulation of C18 polyunsaturated fatty acids (PUFAs) leading to viral proliferation and symptom development. The overexpression of one of the two novel LD-associated proteins (LDAPs) of maize (ZmLDAP1 and ZmLDAP2) induces LD clustering. The core capsid protein P8 of RBSDV interacts with ZmLDAP2 and prevents its degradation through the ubiquitin-proteasome system mediated by a UBX domain-containing protein, PUX10. In addition, silencing of ZmLDAP2 downregulates the expression of FA desaturase genes in maize, leading to a decrease in C18 PUFAs levels and suppression of RBSDV accumulation. Our findings reveal that plant virus may recruit LDAP to regulate cellular FA metabolism to promote viral multiplication and infection. These results expand the knowledge of LD functions and viral infection mechanisms in plants., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Maize rough dwarf disease (MRDD) threatens maize production globally. The P7-1 effector of the rice black-streaked dwarf virus (RBSDV) targets maize Rab GDP dissociation inhibitor alpha (ZmGDIα) to cause MRDD. However, P7-1 has difficulty recruiting a ZmGDIα variant with an alternative helitron-derived exon 10 (ZmGDIα-hel), resulting in recessive resistance. Here, we demonstrate that P7-1 can recruit another maize protein, gibberellin 2-oxidase 13 (ZmGA2ox7.3), which also exhibits tighter binding affinity for ZmGDIα than ZmGDIα-hel. The oligomerization of ZmGA2ox7.3 is vital for its function in converting bioactive gibberellins into inactive forms. Moreover, the enzymatic activity of ZmGA2ox7.3 oligomers increases when forming hetero-oligomers with P7-1/ZmGDIα, but decreases when ZmGDIα-hel replaces ZmGDIα. Viral infection significantly promotes ZmGA2ox7.3 expression and oligomerization in ZmGDIα-containing susceptible maize, resulting in reduced bioactive GA 1 /GA 4 levels. This causes an auxin/cytokinin imbalance and ultimately manifests as MRDD syndrome. Conversely, in resistant maize, ZmGDIα-hel counters these virus-induced changes, thereby mitigating MRDD severity., (© 2024. The Author(s).)

The eukaryotic mRNA surveillance pathway, a pivotal guardian of mRNA fidelity, stands at the nexus of diverse biological processes, including antiviral immunity. Despite the recognized function of splicing factors on mRNA fate, the intricate interplay shaping the mRNA surveillance pathway remains elusive. We illustrate that the conserved splicing factor U2 snRNP auxiliary factor large subunit B (U2AF65B) modulates splicing of mRNA surveillance complex, contributing to transcriptomic homeostasis in maize. The functionality of the mRNA surveillance pathway requires ZmU2AF65B-mediated normal splicing of upstream frameshift 3 ( ZmUPF3 ) pre-mRNA, encoding a core factor in this pathway. Intriguingly, sugarcane mosaic virus (SCMV)-coded nuclear inclusion protein a protease (NIa-Pro) hinders the splicing function of ZmU2AF65B. Furthermore, NIa-Pro disrupts ZmU2AF65B binding to ZmUPF3 pre-mRNA, leading to dysregulated splicing of ZmUPF3 transcripts and, consequently, impairing mRNA surveillance, thus facilitating viral infection. Together, this study establishes that splicing governs the mRNA surveillance pathway and identifies a pathogenic protein capable of disrupting this regulation to compromise RNA immunity.

Exportin 1 (XPO1) is the major karyopherin-β nuclear receptor mediating the nuclear export of hundreds of proteins and some classes of RNA and regulates several critical processes in the cell, including cell-cycle progression, transcription and translation. Viruses have co-opted XPO1 to promote nucleocytoplasmic transport of viral proteins and RNA. Maize mosaic virus (MMV) is a plant-infecting rhabdovirus transmitted in a circulative propagative manner by the corn planthopper, Peregrinus maidis. MMV replicates in the nucleus of plant and insect hosts, and it remains unknown whether MMV co-opts P. maidis XPO1 (PmXPO1) to complete its life cycle. Because XPO1 plays multiple regulatory roles in cell functions and virus infection, we hypothesized that RNAi-mediated silencing of XPO1 would negatively affect MMV accumulation and insect physiology. Although PmXPO1 expression was not modulated during MMV infection, PmXPO1 knockdown negatively affected MMV accumulation in P. maidis at 12 and 15 days after microinjection. Likewise, PmXPO1 knockdown negatively affected P. maidis survival and reproduction. PmXPO1 exhibited tissue-specific expression patterns with higher expression in the ovaries compared with the guts of adult females. Survival rate was significantly lower for PmXPO1 knockdown females, compared with controls, but no effect was observed for males. PmXPO1 knockdown experiments revealed a role for PmXPO1 in ovary function and egg production. Oviposition and egg hatch on plants were dramatically reduced in females treated with dsRNA PmXPO1. These results suggest that PmXPO1 is a positive regulator of P. maidis reproduction and that it plays a proviral role in the insect vector supporting MMV infection., (© 2024 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Rice black-streaked dwarf virus (RBSDV) is an etiological agent of a destructive disease infecting some economically important crops from the Gramineae family in Asia. While RBSDV causes high yield losses, genetic characteristics of replicative viral populations have not been investigated within different host plants and insect vectors. Herein, eleven publicly available RNA-Seq datasets from Chinese RBSDV-infected rice, maize, and viruliferous planthopper (Laodelphax striatellus) were obtained from the NCBI database. The patterns of SNP and RNA expression profiles of expected RBSDV populations were analyzed by CLC Workbench 20 and Geneious Prime software. These analyses discovered 2,646 mutations with codon changes in RBSDV whole transcriptome and forty-seven co-mutated hotspots with high variant frequency within the crucial regions of S5-1, S5-2, S6, S7-1, S7-2, S9, and S10 open reading frames (ORFs) which are responsible for some virulence and host range functions. Moreover, three joint mutations are located on the three-dimensional protein of P9-1. The infected RBSDV-susceptible rice cultivar KTWYJ3 and indigenous planthopper datasets showed more co-mutated hotspot numbers than others. Our analyses showed the expression patterns of viral genomic fragments varied depending on the host type. Unlike planthopper, S5-1, S2, S6, and S9-1 ORFs, respectively had the greatest read numbers in host plants; and S5-2, S9-2, and S7-2 were expressed in the lowest level. These findings underscore virus/host complexes are effective in the genetic variations and gene expression profiles of plant viruses. Our analysis revealed no evidence of recombination events. Interestingly, the negative selection was observed at 12 RBSDV ORFs, except for position 1015 in the P1 protein, where a positive selection was detected. The research highlights the potential of SRA datasets for analysis of the virus cycle and enhances our understanding of RBSDV's genetic diversity and host specificity., (© 2024. The Author(s).)

To describe the transmission dynamics of maize streak virus infection, in the paper, we first formulate a stochastic maize streak virus infection model, in which the stochastic fluctuations are depicted by a logarithmic Ornstein-Uhlenbeck process. This approach is reasonable to simulate the random impacts of main parameters both from the biological significance and the mathematical perspective. Then we investigate the detailed dynamics of the stochastic system, including the existence and uniqueness of the global solution, the existence of a stationary distribution, the exponential extinction of the infected maize and infected leafhopper vector. Especially, by solving the five-dimensional algebraic equations corresponding to the stochastic system, we obtain the specific expression of the probability density function near the quasi-endemic equilibrium of the stochastic system, which provides valuable insights into the stationary distribution. Finally, the model is discretized using the Milstein higher-order numerical method to illustrate our theoretical results numerically. Our findings provide a groundwork for better methods of preventing the spread of this type of virus., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In maize, two pyruvate orthophosphate dikinase (PPDK) regulatory proteins, ZmPDRP1 and ZmPDRP2, are respectively specific to the chloroplast of mesophyll cells (MCs) and bundle sheath cells (BSCs). Functionally, ZmPDRP1/2 catalyse both phosphorylation/inactivation and dephosphorylation/activation of ZmPPDK, which is implicated as a major rate-limiting enzyme in C4 photosynthesis of maize. Our study here showed that maize plants lacking ZmPDRP1 or silencing of ZmPDRP1/2 confer resistance to a prevalent potyvirus sugarcane mosaic virus (SCMV). We verified that the C-terminal domain (CTD) of ZmPDRP1 plays a key role in promoting viral infection while independent of enzyme activity. Intriguingly, ZmPDRP1 and ZmPDRP2 re-localize to cytoplasmic viral replication complexes (VRCs) following SCMV infection. We identified that SCMV-encoded cytoplasmic inclusions protein CI targets directly ZmPDRP1 or ZmPDRP2 or their CTDs, leading to their re-localization to cytoplasmic VRCs. Moreover, we found that CI could be degraded by the 26S proteasome system, while ZmPDRP1 and ZmPDRP2 could up-regulate the accumulation level of CI through their CTDs by a yet unknown mechanism. Most importantly, with genetic, cell biological and biochemical approaches, we provide evidence that BSCs-specific ZmPDRP2 could accumulate in MCs of Zmpdrp1 knockout (KO) lines, revealing a unique regulatory mechanism crossing different cell types to maintain balanced ZmPPDK phosphorylation, thereby to keep maize normal growth. Together, our findings uncover the genetic link of the two cell-specific maize PDRPs, both of which are co-opted to VRCs to promote viral protein accumulation for robust virus infection., (© 2024 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Barley yellow dwarf (BYD) is one of the economically most important virus diseases of cereals worldwide, causing yield losses up to 80%. The means to control BYD are limited, and the use of genetically resistant cultivars is the most economical and environmentally friendly approach. The objectives of this study were i) to identify the causative gene for BYD virus (BYDV)-PAV resistance in maize, ii) to identify single nucleotide polymorphisms and/or structural variations in the gene sequences, which may cause differing susceptibilities to BYDV-PAV of maize inbreds, and iii) to characterize the effect of BYDV-PAV infection on gene expression of susceptible, tolerant, and resistant maize inbreds. Using two biparental mapping populations, we could reduce a previously published quantitative trait locus for BYDV-PAV resistance in maize to ~ 0.3 Mbp, comprising nine genes. Association mapping and gene expression analysis further reduced the number of candidate genes for BYDV-PAV resistance in maize to two: Zm00001eb428010 and Zm00001eb428020. The predicted functions of these genes suggest that they confer BYDV-PAV resistance either via interfering with virus replication or by inducing reactive oxygen species signaling. The gene sequence of Zm00001eb428010 is affected by a 54 bp deletion in the 5`-UTR and a protein altering variant in BYDV-PAV-resistant maize inbreds but not in BYDV-PAV-susceptible and -tolerant inbreds. This finding suggests that altered abundance and/or properties of the proteins encoded by Zm00001eb428010 may lead to BYDV-PAV resistance., (© 2024. The Author(s).)

Maize dwarf mosaic (MDM) is one of the most important virus diseases of maize worldwide. Caused by the potyviruses maize dwarf mosaic virus (MDMV) or sugarcane mosaic virus (SCMV), MDM can cause up to 90% yield loss in susceptible hybrids. One of the most effective management strategies for MDM is growing potyvirus-resistant corn varieties. However, yield impacts associated with MDM and the corresponding efficacy of genetic resistance present in modern United States commercial hybrid lines is uncharacterized. In this study, we evaluated the disease response of 78 commercial hybrids to MDMV and SCMV and quantified yield losses associated with infection over multiple trials. We determined that while 97% of the hybrids tested were resistant to MDMV, 100% were susceptible to SCMV, with mean disease incidence per line averaging between 45 and 78% across six trial years. Despite only one hybrid displaying visible mosaic symptoms when inoculated with MDMV, MDMV reduced average yields by approximately 5% across all hybrids compared with the mock-inoculated treatment. The yield impact of SCMV was more severe, reducing average yields by 10% across replicated experiments. These results indicate that while most commercial hybrids are resistant to MDMV, possibly due to the presence of the major Scmv1 resistance locus on chromosome 6, additional potyvirus resistance genes are needed to manage SCMV-induced MDM. Pyramiding resistance loci, such as Scmv2 on chromosome 3 or Scmv3 on chromosome 10 in addition to Scmv1 , could be an effective strategy to mitigate the yield impact of MDM disease., Competing Interests: The author(s) declare no conflict of interest.

Maize rough dwarf disease (MRDD) threatens the sustainable production of major cereal crops. Recently, Xu et al. reported a new resistance gene, ZmGLK36, which promotes MRDD resistance in maize by increasing jasmonic acid (JA)-mediated defence. This discovery provides opportunities to develop resistance to rice black-streaked dwarf virus (RBSDV) in other cereal crops such as rice and wheat., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Maize yellow mosaic virus (MaYMV) is an emerging polerovirus that has been detected in maize, other cereal crops, and weedy grass species in Asia, Africa, and the Americas. Disease symptoms in maize include prominent leaf tip reddening and stunting. Infection by MaYMV has been reported to reduce plant growth and yields by 10 to 30% in some instances. In this study, an experimental host range for MaYMV among agronomically important cereal crops and common grasses was established. Additional aphid species were assessed as potential vectors for MaYMV, and their transmission efficiencies were determined. Here, we report oats, foxtail millet, barley, and rye as new experimental cereal crop hosts of MaYMV in addition to confirming the previously reported hosts of corn, sorghum, wheat, and broom millet. Four of the nine other grass species evaluated were also identified as suitable experimental hosts for MaYMV: ryegrass, switchgrass, green foxtail, and sand love grass. Interestingly, no visible symptoms were present in any of the infected hosts besides the susceptible maize control. Vector range studies identified the greenbug aphid Schizaphis graminum as a new vector of MaYMV, though transmission efficiency was lower than the previously reported Rhopalosiphum maidis vector and similar to the other known aphid vector R. padi . Given MaYMV's global ubiquity, ability to evade detection, and broad host range, further characterization of yield impacts and identification of viable control strategies are desirable., Competing Interests: The author(s) declare no conflict of interest.

MicroRNAs (miRNAs) are widely involved in various biological processes of plants and contribute to plant resistance against various pathogens. In this study, upon sugarcane mosaic virus (SCMV) infection, the accumulation of maize (Zea mays) miR398b (ZmmiR398b) was significantly reduced in resistant inbred line Chang7-2, while it was increased in susceptible inbred line Mo17. Degradome sequencing analysis coupled with transient co-expression assays revealed that ZmmiR398b can target Cu/Zn-superoxidase dismutase2 (ZmCSD2), ZmCSD4, and ZmCSD9 in vivo, of which the expression levels were all upregulated by SCMV infection in Chang7-2 and Mo17. Moreover, overexpressing ZmmiR398b (OE398b) exhibited increased susceptibility to SCMV infection, probably by increasing reactive oxygen species (ROS) accumulation, which were consistent with ZmCSD2/4/9-silenced maize plants. By contrast, silencing ZmmiR398b (STTM398b) through short tandem target mimic (STTM) technology enhanced maize resistance to SCMV infection and decreased ROS levels. Interestingly, copper (Cu)-gradient hydroponic experiments demonstrated that Cu deficiency promoted SCMV infection while Cu sufficiency inhibited SCMV infection by regulating accumulations of ZmmiR398b and ZmCSD2/4/9 in maize. These results revealed that manipulating the ZmmiR398b-ZmCSD2/4/9-ROS module provides a prospective strategy for developing SCMV-tolerant maize varieties., (© 2024 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Maize lethal necrosis is a destructive virus disease of maize caused by maize chlorotic mottle virus (MCMV) in combination with a virus in the family Potyviridae. Emergence of MLN is typically associated with the introduction of MCMV or its vectors and understanding its spread through seed is critical for disease management. Previous studies suggest that although MCMV is detected on seed, the seed transmission rate of this virus is low. However, mechanisms influencing its transmission are poorly understood. Elucidating these mechanisms is crucial for informing strategies to prevent spread on contaminated seed. In this study, we evaluated the rate of MCMV seed transmission using seed collected from plants that were artificially inoculated with MCMV isolates from Hawaii and Kenya. Grow-out tests indicated that MCMV transmission through seed was rare, with a rate of 0.004% among the more than 85,000 seed evaluated, despite detection of MCMV at high levels in the seed lots. To understand factors that limit transmission from seed, MCMV distribution in seed tissues was examined using serology and immunolocalization. The virus was present at high levels in maternal tissues, the pericarp and pedicel, but absent from filial endosperm and embryo seed tissues. The ability to transmit MCMV from seed to uninfected plants was tested to evaluate virus viability. Transmission was negatively associated with both seed maturity and moisture content. Transmission of MCMV from infested seed dried to less than 15% moisture was not detected, suggesting proper handling could be important for minimizing spread of MCMV through seed., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Maize chlorotic dwarf virus (MCDV) encodes a 3C-like protease that cleaves the N-terminal polyprotein (R78) as previously demonstrated. Here, we examined amino acid residues required for catalytic activity of the protease, including those in the predicted catalytic triad, amino acid residues H2667, D2704, and C2798, as well as H2817 hypothesized to be important in substrate binding. These and other residues were targeted for mutagenesis and tested for proteolytic cleavage activity on the N-terminal 78 kDa MCDV-S polyprotein substrate to identify mutants that abolished catalytic activity. Mutations that altered the predicted catalytic triad residues and H2817 disrupted MCDV-S protease activity, as did mutagenesis of a conserved tyrosine residue, Y2774. The protease activity and R78 cleavage of orthologs from divergent MCDV isolates MCDV-Tn and MCDV-M1, and other waikavirus species including rice tungro spherical virus (RTSV) and bellflower vein chlorosis virus (BVCV) were also examined., (Published by Elsevier Inc.)

Maize rough dwarf disease (MRDD) is a viral disease that causes substantial yield loss, especially in China's summer planted maize area. Discovery of resistance genes would help in developing high-yielding resistant maize hybrids. Genome-wide association studies (GWASs) have advanced quickly and are now a powerful tool for dissecting complex genetic architectures. In this study, the disease severity index (DSI) of 292 maize inbred lines and an F 6 linkage population were investigated across multiple environments for two years. Using the genotypes obtained from the Maize SNP 50K chip, a GWAS was performed with four analytical models. The results showed that 22 SNPs distributed on chromosomes 1, 3, 4, 6, 7 and 8 were significantly associated with resistance to MRDD (P<0.0001). The SNPs on chromosomes 3, 6 and 8 were consistent with the quantitative trait locus (QTL) regions from linkage mapping in an RIL population. Candidate genes identified by GWAS included an LRR receptor-like serine/threonine-protein kinase (GRMZM2G141288), and a DRE-binding protein (GRMZM2G006745). In addition, we performed an allele variation analysis of the SNP loci selected by GWAS and linkage mapping and found that the main alleles of the two SNP loci PZE&#95;101170408 and PZE&#95;106082685 on chromosome 1 differed in terms of disease-resistant materials and disease-susceptible materials. The identified SNPs and genes provide useful information for MRDD-related gene cloning and insights on the underlying disease resistance mechanisms, and they can be used in marker-assisted breeding to develop MRDD-resistant maize., (Copyright © 2021. Published by Elsevier B.V.)

Sugarcane mosaic virus (SCMV), which belongs to the Potyvirus genus of the family Potyviridae, causes mosaic diseases in canna, sugarcane and maize worldwide. Previously, the genetic variations, timescale, codon usage patterns and host adaptions of SCMV were determined. However, the dinucleotide composition and the dinucleotide bias from hosts or the protein coding regions of the virus have yet to be investigated. In this study, comprehensive analyses of the dinucleotide composition and dinucleotide bias from hosts, lineages and protein coding regions of SCMV were performed using 131 complete genomic sequences. We found that UpG and CpA were largely overrepresented while UpA, CpC, and CpG were largely underrepresented in the polyprotein and 11 protein coding region data sets. SCMV dinucleotide composition bias is more strongly dependent on the protein coding regions than on hosts. A weak association between the dinucleotide composition and SCMV lineages was also observed. Our analysis provides a novel perspective on the molecular evolutionary mechanisms of SCMV and may provide a better understanding of future research on the origin and evolutionary patterns of SCMV., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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&#95;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.

The corn leafhopper (Dalbulus maidis) is an important vector of maize rayado fino virus (MRFV), a positive-strand RNA (+ssRNA) marafivirus which it transmits in a persistent propagative manner. The interaction of D. maidis with MRFV, including infection of the insect and subsequent transmission to new plants, is not well understood at the molecular level. To examine the leafhopper-virus interaction, a D. maidis transcriptome was assembled and differences in transcript abundance between virus-exposed and naive D. maidis were examined at two time points (4 h and 7 days) post exposure to MRFV. The D. maidis transcriptome contained 56,116 transcripts generated from 1,727,369,026 100-nt paired-end reads from whole adult insects. The transcriptome of D. maidis shared highest identity and most orthologs with the leafhopper Graminella nigrifrons (65% of transcripts had matches with E values of <10 -5 ) versus planthoppers Sogatella furcifera (with 23% of transcript matches below the E value cutoff) and Peregrinus maidis (with 21% transcript matches below the E value cutoff), as expected based on taxonomy. D. maidis expressed genes in the Toll, Imd, and Jak/Stat insect immune signaling pathways, RNA interference (RNAi) pathway genes, prophenoloxidase-activating system pathways, and immune recognition protein-encoding genes such as peptidoglycan recognition proteins (PGRPs), antimicrobial peptides, and other effectors. Statistical analysis (performed by R package DESeq2) identified 72 transcripts at 4 h and 67 at 7 days that were significantly responsive to MRFV exposure. Genes expected to be favorable for virus propagation, such as protein synthesis-related genes and genes encoding superoxide dismutase, were significantly upregulated after MRFV exposure. IMPORTANCE The transcriptome of the corn leafhopper, D. maidis , revealed conserved biochemical pathways for immunity and discovered transcripts responsive to MRFV-infected plants at two time points, providing a basis for functional identification of genes that either limit or promote the virus-vector interaction. Compared to other hopper species and the propagative plant viruses they transmit, D. maidis shared 15 responsive transcripts with S. furcifera (to southern rice black-streaked dwarf virus [SRBSDV]), one with G. nigrifrons (to maize fine streak virus [MFSV]), and one with P. maidis (to maize mosaic virus [MMV]), but no virus-responsive transcripts identified were shared among all four hopper vector species.

Virus-induced gene silencing (VIGS) is a versatile and attractive approach for functional gene characterization in plants. Although several VIGS vectors for maize (Zea mays) have been previously developed, their utilities are limited due to low viral infection efficiency, insert instability, short maintenance of silencing, inadequate inoculation method, or abnormal requirement of growth temperature. Here, we established a Cucumber mosaic virus (CMV)-based VIGS system for efficient maize gene silencing that overcomes many limitations of VIGS currently available for maize. Using two distinct strains, CMV-ZMBJ and CMV-Fny, we generated a pseudorecombinant-chimeric (Pr) CMV. Pr CMV showed high infection efficacy but mild viral symptoms in maize. We then constructed Pr CMV-based vectors for VIGS, dubbed Pr CMV VIGS. Pr CMV VIGS is simply performed by mechanical inoculation of young maize leaves with saps of Pr CMV-infected Nicotiana benthamiana under normal growth conditions. Indeed, suppression of isopentenyl/dimethylallyl diphosphate synthase (ZmIspH) expression by Pr CMV VIGS resulted in non-inoculated leaf bleaching as early as 5 d post-inoculation (dpi) and exhibited constant and efficient systemic silencing over the whole maize growth period up to 105 dpi. Furthermore, utilizing a ligation-independent cloning (LIC) strategy, we developed a modified Pr CMV-LIC VIGS vector, allowing easy gene cloning for high-throughput silencing in maize. Thus, our Pr CMV VIGS system provides a much-improved toolbox to facilitate efficient and long-duration gene silencing for large-scale functional genomics in maize, and our pseudorecombination-chimera combination strategy provides an approach to construct efficient VIGS systems in plants., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The economic importance of the maize streak virus disease to the African maize production dynamic is to be appreciated now more than ever due to the preponderant influence of a changing climate. Continued dependence on a single major-effect quantitative trait locus (QTL) called Msv1 on Chromosome 1 of Maize (Zea mays L.) is not guaranteed to ensure durable resistance to the causal pathogen. With over ten decades of research on the disease and its associated host plant resistance mechanisms, it is pertinent to consider future approaches to attaining durability by looking to the synergistic roles of moderate- and minor-effect QTLs located on other chromosomes so as to facilitate a secure farming system for sub-Saharan Africa. For this review, more than 40 publications relating to maize streak disease research were methodically analysed with about 30% making specific reference to conventional, molecular and transgenic approaches employed in introgressing, maintaining and improving streak resistance in maize. A meta-analysis of mapped QTLs conferring streak resistance was conducted in a bid to reveal any inter-dependence or co-localization of resistant loci and to aid decision-making for marker-assisted breeding. With the changing climatic conditions around the globe, man's preparedness in the event of an epidemic following any evolutionary process in the streak viral genome was determined as insufficient. Modern breeding approaches including gene pyramiding that could be considered in maize breeding programmes to ensure durability for streak resistance were proposed while improving maize for other abiotic stress tolerance, particularly drought., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21-99.98) and specificity (100%, 95% CI 96.31-100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation., (© 2021. The Author(s).)

A maize-infecting polerovirus, variously named maize yellow dwarf virus RMV2 (MYDV RMV2), MYDV-like, and maize yellow mosaic virus (MaYMV), is frequently found in mixed infections in plants also infected with maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV), known to synergistically cause maize lethal necrosis (MLN). MaYMV was discovered in deep sequencing studies precipitated by recent MLN emergence and is prevalent at global locations with MLN, but its role in or contribution to disease was not known. We examined how MaYMV impacted disease development in mixed infections with MCMV, SCMV, and both MCMV and SCMV compared with mock-inoculated plants. Results demonstrated that MaYMV symptoms included stunting as well as leaf reddening in single and mixed infections. MaYMV did not recapitulate MLN synergistic disease in double infections in which either MCMV or SCMV was missing (MaYMV + MCMV or MaYMV + SCMV), but did significantly enhance stunting in mixed infections and suppressed titers of both MCMV and SCMV in double infections. Interestingly, MaYMV strongly suppressed the SCMV-induced titer increase of MCMV in triple infections, but MLN symptoms still occurred with the reduced MCMV titer. These data indicate the potential disease impact of this newly discovered ubiquitous maize virus, alone and in the context of MLN.

Background: Maize rough dwarf disease (MRDD), a widespread disease caused by four pathogenic viruses, severely reduces maize yield and grain quality. Resistance against MRDD is a complex trait that controlled by many quantitative trait loci (QTL) and easily influenced by environmental conditions. So far, many studies have reported numbers of resistant QTL, however, only one QTL have been cloned, so it is especially important to map and clone more genes that confer resistance to MRDD., Results: In the study, a major quantitative trait locus (QTL) qMrdd2, which confers resistance to MRDD, was identified and fine mapped. qMrdd2, located on chromosome 2, was consistently identified in a 15-Mb interval between the simple sequence repeat (SSR) markers D184 and D1600 by using a recombinant inbred line (RIL) population derived from a cross between resistant ("80007") and susceptible ("80044") inbred lines. Using a recombinant-derived progeny test strategy, qMrdd2 was delineated to an interval of 577 kb flanked by markers N31 and N42. We further demonstrated that qMrdd2 is an incompletely dominant resistance locus for MRDD that reduced the disease severity index by 20.4%., Conclusions: A major resistance QTL (qMrdd2) have been identified and successfully refined into 577 kb region. This locus will be valuable for improving maize variety resistance to MRDD via marker-assisted selection (MAS).

The distribution and use of pathogen-free planting material ("clean seeds") is a promising method to control plant diseases in developing countries. We address the question of minimizing disease prevalence in plants through the optimal usage of clean seeds. We consider the simplest possible S-I model together with a simple economic criterion to be maximized. The static optimization problem shows a diversity of possible outcomes depending on economical and epidemiological parameters. We derive a simple condition showing to what extent subsidizing clean seeds relative to the epidemiological features of the disease may help eradicate or control the disease. Then we consider dynamic optimal control and Pontryagin's maximum principle to study the optimal usage of clean seeds to control the disease. The dynamical results are comparable to the static ones and are even simpler in some sense. In particular, the condition on the critical subsidy rate that makes clean seed usage economically viable is unchanged from the static optimization case. We discuss how these results may apply to the control of maize lethal necrosis in East-Africa.

Maize streak disease (MSD) is one of the most significant biotic constraints on the production of Africa's most important cereal crop. Until recently, the only virus known to cause severe MSD was the A-strain of maize streak virus (MSV/A), a member of the genus Mastrevirus, family Geminiviridae. However, over the past decade, two other mastreviruses, MSV/C and maize streak Réunion virus (MSRV), have been repeatedly found in the absence of MSV/A in maize plants displaying severe MSD symptoms. Here, we report on infectious clones of MSV/C and MSRV and test their ability to cause severe MSD symptoms. Although cloned MSV/C and MSRV genomes could cause systemic symptomatic infections in MSD-sensitive maize genotypes, these infections yielded substantially milder symptoms than those observed in the field. The MSV/C and MSRV isolates that we have examined are therefore unlikely to cause severe MSD on their own. Furthermore, mixed infections of MSRV and MSV/C with other mild MSV strains also consistently yielded mild MSD symptoms. It is noteworthy that MSRV produces distinctive striate symptoms in maize that are similar in pattern, albeit not in severity, to those seen in the field, showing that this virus may contribute to the severe MSD symptoms seen in the field. Therefore, despite not fulfilling Koch's postulates for MSV/C and MSRV as causal agents of severe MSD, we cannot exclude the possibility that these viruses could be contributing to currently emerging maize diseases.

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22) or phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.

Maize rough dwarf virus (MRDV) is one of the main yield-limiting factors of maize in the Mediterranean. However, knowledge about the interactions between the agroecosystem and the virus-vector-host relationship continues to be limited. We used multi-model inference to test a landscape-scale approach together with variables measured in the field, and we estimated the effects of early and late planting on MRDV incidence. The results revealed that the virus incidence increased by 3% when the planting was delayed, and this increase was coincident with the first peak of the vector population. The variables at the field and landscape scales with a strong effect on virus incidence were the proportions of grasses in adjacent crops, in uncultivated areas, and in edges close to maize plants. Grass plant cover in the edges also affected virus incidence, but these effects varied with the planting period. These findings provide new insights into the causes of MRDV incidence and may provide some guidance to growers to reduce losses caused by the virus. Among the recommendations to be prioritized are early planting, management of grasses at field edges, and non-overlapping cultivation of maize and winter cereals in the same area.

Rice black streaked dwarf virus (RBSDV) is an important agent causing maize rough dwarf disease, whereas the host factors responding to RBSDV infection are poorly understood. To uncover the molecular interactions between RBSDV and maize, a yeast two-hybrid screen of a maize cDNA library was carried out using the viral P8 protein as a bait. ZmAKINβγ-1 and ZmAKINβγ-2 (βγ subunit of Arabidopsis SNF1 kinase homolog in maize) possessing high sequence similarities (encoded by two gene copies) were identified as interaction partners. Their interactions with P8 were confirmed in both Nicotiana benthamiana cells and maize protoplasts by bimolecular fluorescence complementation assay. The accumulation levels of ZmAKINβγ mRNAs were upregulated at the stage of the viral symptoms beginning to appear and then downregulated. ZmAKINβγs are putative regulatory subunits of the SnRK1 complex, a core regulator for energy homeostasis. Knockdown of ZmAKINβγ s in maize regulated the expression levels of the genes involved in sugar synthesis or degradation, and also the contents of both glucose and sucrose. Importantly, downregulation of ZmAKINβγ s expressions facilitated the accumulation of RBSDV in maize. These results implicate a role of ZmAKINβγ s in the regulation of primary carbohydrate metabolism, and in the defense against RBSDV infection.

Plant disease resistance proteins (R-proteins) detect specific pathogen-derived molecules, triggering a defence response often including a rapid localized cell death at the point of pathogen penetration called the hypersensitive response (HR). The maize Rp1-D21 gene encodes a protein that triggers a spontaneous HR causing spots on leaves in the absence of any pathogen. Previously, we used fine mapping and functional analysis in a Nicotiana benthamiana transient expression system to identify and characterize a number of genes associated with variation in Rp1-D21-induced HR. Here we describe a system for characterizing genes mediating HR, using virus-induced gene silencing (VIGS) in a maize line carrying Rp1-D21. We assess the roles of 12 candidate genes. Three of these genes, SGT1, RAR1, and HSP90, are required for HR induced by a number of R-proteins across several plant-pathogen systems. We confirmed that maize HSP90 was required for full Rp1-D21-induced HR. However, suppression of SGT1 expression unexpectedly increased the severity of Rp1-D21-induced HR while suppression of RAR1 expression had no measurable effect. We confirmed the effects on HR of two genes we had previously validated in the N. benthamiana system, hydroxycinnamoyltransferase and caffeoyl CoA O-methyltransferase. We further showed the suppression the expression of two previously uncharacterized, candidate genes, IQ calmodulin binding protein (IQM3) and vacuolar protein sorting protein 37, suppressed Rp1-D21-induced HR. This approach is an efficient way to characterize the roles of genes modulating the hypersensitive defence response and other dominant lesion phenotypes in maize., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Maize streak virus disease (MSVD), caused by Maize streak virus (MSV; genus Mastrevirus), is one of the most severe and widespread viral diseases that adversely reduces maize yield and threatens food security in Africa. An effective control and management of MSVD requires robust and sensitive diagnostic tests capable of rapid detection of MSV. In this study, a loop-mediated isothermal amplification (LAMP) assay was designed for the specific detection of MSV. This test has shown to be highly specific and reproducible and able to detect MSV in as little as 10 fg/µl of purified genomic DNA obtained from a MSV-infected maize plant, a sensitivity 10 5 times higher to that obtained with polymerase chain reaction (PCR) in current general use. The high degree of sequence identity between Zambian and other African MSV isolates indicate that this LAMP assay can be used for detecting MSV in maize samples from any region in Africa. Furthermore, this assay can be adopted in minimally equipped laboratories and with potential use in plant clinic laboratories across Africa strengthening diagnostic capacity in countries dealing with MSD.

The COVID-19 pandemic has shown that understanding the genomics of a virus, diagnostics and breaking virus transmission is essential in managing viral pandemics. The same lessons can apply for plant viruses. There are plant viruses that have severely disrupted crop production in multiple countries, as recently seen with maize lethal necrosis disease in eastern and southern Africa. High-throughput sequencing (HTS) is needed to detect new viral threats. Equally important is building local capacity to develop the tools required for rapid diagnosis of plant viruses. Most plant viruses are insect-vectored, hence, biological insights on virus transmission are vital in modelling disease spread. Research in Africa in these three areas is in its infancy and disjointed. Despite intense interest, uptake of HTS by African researchers is hampered by infrastructural gaps. The use of whole-genome information to develop field-deployable diagnostics on the continent is virtually inexistent. There is fledgling research into plant-virus-vector interactions to inform modelling of viral transmission. The gains so far have been modest but encouraging, and therefore must be consolidated. For this, I propose the creation of a new Research Centre for Africa. This bold investment is needed to secure the future of Africa's crops from insect-vectored viral diseases.

Pathogens disturb alternative splicing patterns of infected eukaryotic hosts. However, in plants it is unknown if this is incidental to infection or represents a pathogen-induced remodeling of host gene expression needed to support infection. Here, we compared changes in transcription and protein accumulation with changes in transcript splicing patterns in maize ( Zea mays ) infected with the globally important pathogen sugarcane mosaic virus (SCMV). Our results suggested that changes in alternative splicing play a major role in determining virus-induced proteomic changes. Focusing on maize phytoene synthase1 ( ZmPSY1 ), which encodes the key regulatory enzyme in carotenoid biosynthesis, we found that although SCMV infection decreases total ZmPSY1 transcript accumulation, the proportion of splice variant T001 increases by later infection stages so that ZmPSY1 protein levels are maintained. We determined that ZmPSY1 has two leaf-specific transcripts, T001 and T003, distinguished by differences between the respective 3'-untranslated regions (UTRs). The shorter 3'-UTR of T001 makes it the more efficient mRNA. Nonsense ZmPSY1 mutants or virus-induced silencing of ZmPSY1 expression suppressed SCMV accumulation, attenuated symptoms, and decreased chloroplast damage. Thus, ZmPSY1 acts as a proviral host factor that is required for virus accumulation and pathogenesis. Taken together, our findings reveal that SCMV infection-modulated alternative splicing ensures that ZmPSY1 synthesis is sustained during infection, which supports efficient virus infection., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Insect salivary glands play an important role for host feeding, specifically by secreting salivary proteins for digestion and potentially modulating host defenses. Compared to other hemipterans, the significance of salivary glands is less studied in the black-faced leafhopper, Graminella nigrifrons , a crop pest that vectors several agronomically important plant viruses. To identify functionally important genes in the salivary glands of the black-faced leafhopper, we compared transcriptomes between adult salivary glands (SG) and the remaining carcasses. We identified 14,297 salivary gland-enriched transcripts and 195 predicted secretory peptides (i.e . , with a signal peptide and extracellular localization characteristics). Overall, the SG transcriptome included functions such as 'oxidoreduction', 'membrane transport', and 'ATP-binding', which might be important for the fundamental physiology of this tissue. We further evaluated transcripts with potential contributions in host feeding using RT-qPCR. Two SG-enriched transcripts (log 2 fold change > 5), GnP19 and GnE63 (a putative calcium binding protein), were significantly upregulated in maize-fed adults relative to starved adults, validating their importance in feeding. The SG-enriched transcripts of the black-faced leafhopper could play a potential role for interacting with maize and could be targets of interest for further functional studies and improve pest control and disease transmission., Competing Interests: The authors declare no conflict of interest.

Maize chlorotic mottle virus (MCMV) combines with a potyvirus in maize lethal necrosis disease (MLND), a serious emerging disease worldwide. To inform resistance strategies, we characterized the translation initiation mechanism of MCMV. We report that MCMV RNA contains a cap-independent translation element (CITE) in its 3' untranslated region (UTR). The MCMV 3' CITE (MTE) was mapped to nucleotides 4164 to 4333 in the genomic RNA. 2'-Hydroxyl acylation analyzed by primer extension (SHAPE) probing revealed that the MTE is a distinct variant of the panicum mosaic virus-like 3' CITE (PTE). Like the PTE, electrophoretic mobility shift assays (EMSAs) indicated that eukaryotic translation initiation factor 4E (eIF4E) binds the MTE despite the absence of an m 7 GpppN cap structure, which is normally required for eIF4E to bind RNA. Using a luciferase reporter system, mutagenesis to disrupt and restore base pairing revealed that the MTE interacts with the 5' UTRs of both genomic RNA and subgenomic RNA1 via long-distance kissing stem-loop interaction to facilitate translation. The MTE stimulates a relatively low level of translation and has a weak, if any, pseudoknot, which is present in the most active PTEs, mainly because the MTE lacks the pyrimidine-rich tract that base pairs to a G-rich bulge to form the pseudoknot. However, most mutations designed to form a pseudoknot decreased translation activity. Mutations in the viral genome that reduced or restored translation prevented and restored virus replication, respectively, in maize protoplasts and in plants. In summary, the MTE differs from the canonical PTE but falls into a structurally related class of 3' CITEs. IMPORTANCE In the past decade, maize lethal necrosis disease has caused massive crop losses in East Africa. It has also emerged in China and parts of South America. Maize chlorotic mottle virus (MCMV) infection is required for this disease. While some tolerant maize lines have been identified, there are no known resistance genes that confer immunity to MCMV. In order to improve resistance strategies against MCMV, we focused on how the MCMV genome is translated, the first step of gene expression by all positive-strand RNA viruses. We identified a structure (cap-independent translation element) in the 3' untranslated region of the viral RNA genome that allows the virus to usurp a host translation initiation factor, eIF4E, in a way that differs from host mRNA interactions with the translational machinery. This difference indicates eIF4E may be a soft target for engineering of-or breeding for-resistance to MCMV., (Copyright © 2020 Carino et al.)

Maize is the most important food crop in Kenya accounting for more than 51 % of all staples grown in the country. Out of Kenya's 5.3 million ha total crops area, more than 2.1 million ha is occupied by maize which translates to 40 % of all crops area. However, with the emergence of maize lethal necrosis (MLN) disease in 2011, the average yields plummeted to all-time lows with severely affected counties recording 90-100% yield loss in 2013 and 2014. The disease is mainly caused by Maize chlorotic mottle virus (MCMV) in combination with Sugarcane mosaic virus (SCMV) or other potyviruses. In this study, a country-wide survey was carried out to assess the MLN causing viruses in Kenya, their distribution, genetic diversity, and recombination. The causative viruses of MLN were determined by RT-PCR using virus-specific primers and DAS-ELISA. Next-generation sequencing (NGS) data was generated, viral sequences identified, genetic diversity of MLN viruses was determined, and recombination was evaluated. MCMV and SCMV were detected in all the maize growing regions at varying levels of incidence, and severity while MaYMV, a polerovirus was detected in some samples through NGS. However, there were some samples in this study where only MCMV was detected with severe MLN symptoms. SCMV Sequences were highly diverse while MCMV sequences exhibited low variability. Potential recombination events were detected only in SCMV explaining the elevated level of diversity and associated risk of this virus in Kenya and the eastern Africa region., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Nine complete nucleotide sequences of geminialphasatellites (subfamily Geminialphasatellitinae, family Alphasatellitidae) recovered from the wild Poaceae Sorghum arundinaceum collected in Reunion are described and analyzed. While the helper geminivirus was identified as an isolate of maize streak virus (genus Mastrevirus, family Geminiviridae), the geminialphasatellite genomes were most closely related to, and shared ~63% identity with, clecrusatellites. Even though the geminialphasatellite molecules lack an adenine rich-region, they have the typical size of geminialphasatellites, encode a replication-associated protein in the virion sense, and have probable stem-loop structures at their virion-strand origins of replication. According to the proposed geminialphasatellite species and genus demarcation thresholds (88% and 70% nucleotide identity, respectively), the genomes identified here represent a new species (within a new genus) for which we propose the name "Sorghum mastrevirus-associated alphasatellite" (genus "Sorgasalphasatellite").

Maize lethal necrosis (MLN), a complex viral disease, emerged as a serious threat to maize production and the livelihoods of smallholders in eastern Africa since 2011, primarily due to the introduction of maize chlorotic mottle virus (MCMV). The International Maize and Wheat Improvement Center (CIMMYT), in close partnership with national and international partners, implemented a multi-disciplinary and multi-institutional strategy to curb the spread of MLN in sub-Saharan Africa, and mitigate the impact of the disease. The strategy revolved around a) intensive germplasm screening and fast-tracked development and deployment of MLN-tolerant/resistant maize hybrids in Africa-adapted genetic backgrounds; b) optimizing the diagnostic protocols for MLN-causing viruses, especially MCMV, and capacity building of relevant public and private sector institutions on MLN diagnostics and management; c) MLN monitoring and surveillance across sub-Saharan Africa in collaboration with national plant protection organizations (NPPOs); d) partnership with the private seed sector for production and exchange of MLN pathogen-free commercial maize seed; and e) awareness creation among relevant stakeholders about MLN management, including engagement with policy makers. The review concludes by highlighting the need to keep continuous vigil against MLN-causing viruses, and preventing any further spread of the disease to the major maize-growing countries that have not yet reported MLN in sub-Saharan Africa., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Maize rough dwarf disease (MRDD), caused by various species of the genus Fijivirus, threatens maize production worldwide. We previously identified a quantitative locus qMrdd1 conferring recessive resistance to one causal species, rice black-streaked dwarf virus (RBSDV). Here, we show that Rab GDP dissociation inhibitor alpha (RabGDIα) is the host susceptibility factor for RBSDV. The viral P7-1 protein binds tightly to the exon-10 and C-terminal regions of RabGDIα to recruit it for viral infection. Insertion of a helitron transposon into RabGDIα intron 10 creates alternative splicing to replace the wild-type exon 10 with a helitron-derived exon 10. The resultant splicing variant RabGDIα-hel has difficulty being recruited by P7-1, thus leading to quantitative recessive resistance to MRDD. All naturally occurring resistance alleles may have arisen from a recent single helitron insertion event. These resistance alleles are valuable to improve maize resistance to MRDD and potentially to engineer RBSDV resistance in other crops.

For pathogens infecting single host species evolutionary trade-offs have previously been demonstrated between pathogen-induced mortality rates and transmission rates. It remains unclear, however, how such trade-offs impact sub-lethal pathogen-inflicted damage, and whether these trade-offs even occur in broad host-range pathogens. Here, we examine changes over the past 110 years in symptoms induced in maize by the broad host-range pathogen, maize streak virus (MSV). Specifically, we use the quantified symptom intensities of cloned MSV isolates in differentially resistant maize genotypes to phylogenetically infer ancestral symptom intensities and check for phylogenetic signal associated with these symptom intensities. We show that whereas symptoms reflecting harm to the host have remained constant or decreased, there has been an increase in how extensively MSV colonizes the cells upon which transmission vectors feed. This demonstrates an evolutionary trade-off between amounts of pathogen-inflicted harm and how effectively viruses position themselves within plants to enable onward transmission., Competing Interests: AM, SD, PH, KO, BO, MB, DL, RS, LD, SM, ER, AK, EY, PL, RF, PR, DS, GH, MS, PL, AV, DM No competing interests declared, (© 2020, Monjane et al.)

Plant viruses can alter the behavior or performance of their arthropod vectors, either indirectly (through effects of virus infection on the host plant) or directly (from virus acquisition by the vector). Given the diversity of plant viruses and their arthropod vectors, the effects for any specific system are not possible to predict. Here, we present experimental evidence that acquisition of maize Iranian mosaic virus (MIMV, genus Nucleorhabdovirus, family Rhabdoviridae) modifies the biological traits of its insect vector, the small brown planthopper (SBPH) Laodelphax striatellus. MIMV is an economically important virus of maize and several other grass species. It is transmitted by SBPHs in a persistent-propagative manner. We evaluated the effects of MIMV acquisition by SBPH on its life history when reared on healthy barley plants (Hordeum vulgare). We conclude that 1) MIMV acquisition by SBPHs increases female fecundity, duration of the nymph stage, adult longevity, and survival of SBPHs, (2) the mortality rate and female-to-male sex ratio are reduced in MIMV-infected planthoppers, and (3) MIMV infection increases the concentration of some biochemical components of the infected plants, including carbohydrates, some amino acids, and total protein, which might influence the life traits of its insect vector. The results indicate the potential of MIMV to improve the ecological fitness of its vector, SBPH, through direct or indirect effects, with the potential to increase the spread of the virus.

Maize lethal necrosis (MLN) occurs when maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV) co-infect maize plant. Yield loss of up to 100% can be experienced under severe infections. Identification and validation of genomic regions and their flanking markers can facilitate marker assisted breeding for resistance to MLN. To understand the status of previously identified quantitative trait loci (QTL)in diverse genetic background, F 3 progenies derived from seven bi-parental populations were genotyped using 500 selected kompetitive allele specific PCR (KASP) SNPs. The F 3 progenies were evaluated under artificial MLN inoculation for three seasons. Phenotypic analyses revealed significant variability ( P ≤ 0.01) among genotypes for responses to MLN infections, with high heritability estimates (0.62 to 0.82) for MLN disease severity and AUDPC values. Linkage mapping and joint linkage association mapping revealed at least seven major QTL ( qMLN3&#95;130 and qMLN3&#95;142 , qMLN5&#95;190 and qMLN5&#95;202 , qMLN6&#95;85 and qMLN6&#95;157 qMLN8&#95;10 and qMLN9&#95;142 ) spread across the 7-biparetal populations, for resistance to MLN infections and were consistent with those reported previously. The seven QTL appeared to be stable across genetic backgrounds and across environments. Therefore, these QTL could be useful for marker assisted breeding for resistance to MLN.

Maize lethal necrosis (MLN), caused by co-infection of maize chlorotic mottle virus and sugarcane mosaic virus, can lead up to 100% yield loss. Identification and validation of genomic regions can facilitate marker assisted breeding for resistance to MLN. Our objectives were to identify marker-trait associations using genome wide association study and assess the potential of genomic prediction for MLN resistance in a large panel of diverse maize lines. A set of 1400 diverse maize tropical inbred lines were evaluated for their response to MLN under artificial inoculation by measuring disease severity or incidence and area under disease progress curve (AUDPC). All lines were genotyped with genotyping by sequencing (GBS) SNPs. The phenotypic variation was significant for all traits and the heritability estimates were moderate to high. GWAS revealed 32 significantly associated SNPs for MLN resistance (at p < 1.0 × 10 -6 ). For disease severity, these significantly associated SNPs individually explained 3-5% of the total phenotypic variance, whereas for AUDPC they explained 3-12% of the total proportion of phenotypic variance. Most of significant SNPs were consistent with the previous studies and assists to validate and fine map the big quantitative trait locus (QTL) regions into few markers' specific regions. A set of putative candidate genes associated with the significant markers were identified and their functions revealed to be directly or indirectly involved in plant defense responses. Genomic prediction revealed reasonable prediction accuracies. The prediction accuracies significantly increased with increasing marker densities and training population size. These results support that MLN is a complex trait controlled by few major and many minor effect genes.

Maize chlorotic mottle virus (MCMV) has been occurring frequently worldwide and causes severe yield losses in maize ( Zea mays ). To better investigate the destructive effects of MCMV infection on maize plants, isobaric tagging for relative and absolute quantitation (iTRAQ)-based comparative proteomic analysis was performed on MCMV infected maize cv. B73. A total of 972 differentially abundant proteins (DAPs), including 661 proteins with increased abundance and 311 proteins with reduced abundance, were identified in response to MCMV infection. Functional annotations of DAPs and measurement of photosynthetic activity revealed that photosynthesis was decreased, while the abundance of ribosomal proteins, proteins related to stress responses, oxidation-reduction and redox homeostasis was altered significantly during MCMV infection. Two DAPs, disulfide isomerases like protein ZmPDIL-1 and peroxiredoxin family protein ZmPrx5, were further analyzed for their roles during MCMV infection through cucumber mosaic virus-based virus-induced gene silencing (CMV-VIGS). The accumulation of MCMV was suppressed in ZmPDIL-1-silenced or ZmPrx5-silenced B73 maize, suggesting ZmPDIL-1 and ZmPrx5 might enhance host susceptibility to MCMV infection.

Plant RNA virus-based guide RNA (gRNA) delivery has substantial advantages compared to that of the conventional constitutive promoter-driven expression due to the rapid and robust amplification of gRNAs during virus replication and movement. To date, virus-induced genome editing tools have not been developed for wheat and maize. In this study, we engineered a barley stripe mosaic virus (BSMV)-based gRNA delivery system for clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated targeted mutagenesis in wheat and maize. BSMV-based delivery of single gRNAs for targeted mutagenesis was first validated in Nicotiana benthamiana. To extend this work, we transformed wheat and maize with the Cas9 nuclease gene and selected the wheat TaGASR7 and maize ZmTMS5 genes as targets to assess the feasibility and efficiency of BSMV-mediated mutagenesis. Positive targeted mutagenesis of the TaGASR7 and ZmTMS5 genes was achieved for wheat and maize with efficiencies of up to 78% and 48%. Our results provide a useful tool for fast and efficient delivery of gRNAs into economically important crops., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Sugarcane mosaic virus (SCMV) is a pathogen of worldwide importance that causes dwarf mosaic disease on maize (Zea mays). Until now, few maize genes/proteins have been shown to be involved in resistance to SCMV. In this study, we characterized the role of maize phenylalanine ammonia-lyases (ZmPALs) in accumulation of the defence signal salicylic acid (SA) and in resistance to virus infection. SCMV infection significantly increased SA accumulation and expression of SA-responsive pathogenesis-related protein genes (PRs). Interestingly, exogenous SA treatment decreased SCMV accumulation and enhanced resistance. Both reverse transcription-coupled quantitative PCR and RNA-Seq data confirmed that expression levels of at least four ZmPAL genes were significantly up-regulated upon SCMV infection. Knockdown of ZmPAL expression led to enhanced SCMV infection symptom severity and virus multiplication, and simultaneously resulted in decreased SA accumulation and PR gene expression. Intriguingly, application of exogenous SA to SCMV-infected ZmPAL-silenced maize plants decreased SCMV accumulation, showing that ZmPALs are required for SA-mediated resistance to SCMV infection. In addition, lignin measurements and metabolomic analysis showed that ZmPALs are also involved in SCMV-induced lignin accumulation and synthesis of other secondary metabolites via the phenylpropanoid pathway. In summary, our results indicate that ZmPALs are required for SA accumulation in maize and are involved in resistance to virus infection by limiting virus accumulation and moderating symptom severity., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Key Message: Analysis of the genetic architecture of MCMV and MLN resistance in maize doubled-haploid populations revealed QTLs with major effects on chromosomes 3 and 6 that were consistent across genetic backgrounds and environments. Two major-effect QTLs, qMCMV3-108/qMLN3-108 and qMCMV6-17/qMLN6-17, were identified as conferring resistance to both MCMV and MLN. Maize lethal necrosis (MLN) is a serious threat to the food security of maize-growing smallholders in sub-Saharan Africa. The ability of the maize chlorotic mottle virus (MCMV) to interact with other members of the Potyviridae causes severe yield losses in the form of MLN. The objective of the present study was to gain insights and validate the genetic architecture of resistance to MCMV and MLN in maize. We applied linkage mapping to three doubled-haploid populations and a genome-wide association study (GWAS) on 380 diverse maize lines. For all the populations, phenotypic variation for MCMV and MLN was significant, and heritability was moderate to high. Linkage mapping revealed 13 quantitative trait loci (QTLs) for MCMV resistance and 12 QTLs conferring MLN resistance. One major-effect QTL, qMCMV3-108/qMLN3-108, was consistent across populations for both MCMV and MLN resistance. Joint linkage association mapping (JLAM) revealed 18 and 21 main-effect QTLs for MCMV and MLN resistance, respectively. Another major-effect QTL, qMCMV6-17/qMLN6-17, was detected for both MCMV and MLN resistance. The GWAS revealed a total of 54 SNPs (MCMV-13 and MLN-41) significantly associated (P ≤ 5.60 × 10 -05 ) with MCMV and MLN resistance. Most of the GWAS-identified SNPs were within or adjacent to the QTLs detected through linkage mapping. The prediction accuracy for within populations as well as the combined populations is promising; however, the accuracy was low across populations. Overall, MCMV resistance is controlled by a few major and many minor-effect loci and seems more complex than the genetic architecture for MLN resistance.

The corn planthopper, Peregrinus maidis, not only causes direct damage to plants by feeding, but also transmits maize mosaic virus (MMV) to the plant hosts. The virus is transmitted in a propagative manner but the acquisition of MMV by the vector feeding on infected plants can result in low acquisition and inoculation efficiency. Here, we increased the acquisition efficiency by delivering the virus directly into the hemocoel through microinjection, which resulted in efficient virus infection of the insect and transmission to maize. We found that delivery of virus by injection of 10 ng MMV (50 nl, 200 μg/ml virions) into P. maidis resulted in 93% transmission efficiency. In dose-response experiments, MMV abundance in insects and transmission efficiency decreased as the amount of virus inoculum delivered into the hemocoel was reduced. Examination of virus distribution in the vector using immunolabeling and confocal microscopy revealed similar tissue distributions in the injected insects when compared to those of previous studies using feeding on plants for virus acquisition. The utility of virus inoculation by microinjection for functional analysis in virus-vector interaction was explored. Co-microinjection of MMV virions and the dsRNA of PI3Kδ (a transcript that is less abundant in MMV-infected insects), resulted in a reduction in PI3Kδ expression and higher virus titers in P. maidis. These findings demonstrated that virus microinjection is a robust method for obtaining large numbers of infected planthoppers that are competent in transmitting MMV and, in combination with RNAi, could significantly facilitate the functional analysis of P. maidis-MMV interactions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The synergistic infection of maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV) causes maize lethal necrosis, with considerable losses to global maize production. microRNAs (miRNAs) are conserved non-coding small RNAs that play essential regulatory roles in plant development and environmental stress responses, including virus infection. However, the characterization of maize miRNAs in response to synergistic infection of MCMV and SCMV remains largely unknown. In this study, the profiles of small RNAs from MCMV and SCMV single- and co-infected (S + M) maize plants were obtained by high-throughput sequencing. A total of 173 known miRNAs, belonging to 26 miRNA families, and 49 novel miRNAs were profiled. The expression patterns of most miRNAs in S + M-infected maize plants were similar to that in SCMV-infected maize plants, probably due to the existence of RNA silencing suppressor HC-Pro. Northern blotting and quantitative real-time PCR were performed to validate the accumulation of miRNAs and their targets in different experimental treatments, respectively. The down-regulation of miR159, miR393, and miR394 might be involved in antiviral defense to synergistic infection. These results provide novel insights into the regulatory networks of miRNAs in maize plants in response to the synergistic infection of MCMV and SCMV.

RNAtag-seq of maize samples collected in Tanzania revealed the presence of a previously undescribed nucleorhabdovirus, tentatively named "Morogoro maize-associated virus" (MMaV), in three samples. The MMaV genome is 12,185-12,187 nucleotides long and shares a 69-70% nucleotide sequence identity with taro vein chlorosis virus. Annotation of the genomes showed a typical nucleorhabdovirus gene organization. PCR was unable to detect the same virus in the remaining 35 samples collected in the region.