Alan L. Eggenberger

Loss and Gain of Elicitor Function of Soybean Mosaic Virus G7 Provoking Rsv1-Mediated Lethal Systemic Hypersensitive Response Maps to P3

ABSTRACT Rsv1, a single dominant resistance gene in soybean PI 96983 (Rsv1), confers extreme resistance against all known American strains of Soybean mosaic virus (SMV), except G7 and G7d. SMV-G7 provokes a lethal systemic hypersensitive response (LSHR), whereas SMV-G7d, an experimentally evolved variant of SMV-G7, induces systemic mosaic. To identify the elicitor of Rsv1-mediated LSHR, chimeras were constructed by exchanging fragments between the molecularly cloned SMV-G7 (pSMV-G7) and SMV-G7d (pSMV-G7d), and their elicitor functions were assessed on PI 96983 (Rsv1). pSMV-G7-derived chimeras containing only P3 of SMV-G7d lost the elicitor function, while the reciprocal chimera of pSMV-G7d gained the function. The P3 regions of the two viruses differ by six nucleotides, of which two are translationally silent. The four amino acid differences are located at positions 823, 915, 953, and 1112 of the precursor polypeptide. Analyses of the site-directed point mutants of both the viruses revealed that nucleotide substitutions leading to translationally silent mutations as well as reciprocal amino acid substitution at position 915 did not influence the loss or gain of the elicitor function. pSMV-G7-derived mutants with amino acid substitutions at any of the other three positions lost the ability to provoke LSHR but induced SHR instead. Two concomitant amino acid substitutions at positions 823 (V to M) and 953 (K to E) abolished pSMV-G7 elicitor function, provoking Rsv1-mediated SHR. Conversely, pSMV-G7d gained the elicitor function of Rsv1-mediated LSHR by a single amino acid substitution at position 823 (M to V), and mutants with amino acid substitutions at position 953 or 1112 induced SHR instead of mosaic. Taken together, the data suggest that strain-specific P3 of SMV is the elicitor of Rsv1-mediated LSHR.

Evolution of Soybean mosaic virus-G7 molecularly cloned genome in Rsv1-genotype soybean results in emergence of a mutant capable of evading Rsv1-mediated recognition

Plant resistance (R) genes direct recognition of pathogens harboring matching avirluent signals leading to activation of defense responses. It has long been hypothesized that under selection pressure the infidelity of RNA virus replication together with large population size and short generation times results in emergence of mutants capable of evading R-mediated recognition. In this study, the Rsv1/Soybean mosaic virus (SMV) pathosystem was used to investigate this hypothesis. In soybean line PI 96983 (Rsv1), the progeny of molecularly cloned SMV strain G7 (pSMV-G7) provokes a lethal systemic hypersensitive response (LSHR) with up regulation of a defense-associated gene transcript (PR-1). Serial passages of a large population of the progeny in PI 96983 resulted in emergence of a mutant population (vSMV-G7d), incapable of provoking either Rsv1-mediated LSHR or PR-1 protein gene transcript up regulation. An infectious clone of the mutant (pSMV-G7d) was synthesized whose sequences were very similar but not identical to the vSMV-G7d population; however, it displayed a similar phenotype. The genome of pSMV-G7d differs from parental pSMV-G7 by 17 substitutions, of which 10 are translationally silent. The seven amino acid substitutions in deduced sequences of pSMV-G7d differ from that of pSMV-G7 by one each in P1 proteinase, helper component-proteinase, and coat protein, respectively, and by four in P3. To the best of our knowledge, this is the first demonstration in which experimental evolution of a molecularly cloned plant RNA virus resulted in emergence of a mutant capable of evading an R-mediated recognition.

Pathogen-derived transgenic resistance to soybean mosaic virus in soybean

Development of transgenic disease resistance in soybeans, despite progress in other important crop plants, has advanced slowly. In this study, transgenic soybean plants resistant to soybean mosaic virus (SMV) were obtained by transforming with the coat protein gene and the 3′-UTR from SMV. Four insertion events were detected in a T0 plant obtained by using Agrobacterium tumefaciens-mediated transformation. Self-pollination of T0 progeny yielded four homozygous transgenic lines with a single insertion event or combinations of two insertion events in the T3 generation. A single coat protein gene transcript was detected in all four transgenic lines, and virus coat protein was detected in three transgenic lines. Two transgenic lines were highly resistant to the virus. These constitute the first example of stable genetically engineered disease resistance in soybean.

Cytoplasmic inclusion cistron of Soybean mosaic virus serves as a virulence determinant on Rsv3-genotype soybean and a symptom determinant

Soybean mosaic virus (SMV; Potyvirus, Potyviridae) is one of the most widespread viruses of soybean globally. Three dominant resistance genes (Rsv1, Rsv3 and Rsv4) differentially confer resistance against SMV. Rsv1 confers extreme resistance and the resistance mechanism of Rsv4 is associated with late susceptibility. Here, we show that Rsv3 restricts the accumulation of SMV strain G7 to the inoculated leaves, whereas, SMV-N, an isolate of SMV strain G2, establishes systemic infection. This observation suggests that the resistance mechanism of Rsv3 differs phenotypically from those of Rsv1 and Rsv4. To identify virulence determinant(s) of SMV on an Rsv3-genotype soybean, chimeras were constructed by exchanging fragments between avirulent SMV-G7 and the virulent SMV-N. Analyses of the chimeras showed that both the N- and C-terminal regions of the cytoplasmic inclusion (CI) cistron are required for Rsv3-mediated resistance. Interestingly, the N-terminal region of CI is also involved in severe symptom induction in soybean.

Adaptation of Soybean mosaic virus Avirulent Chimeras Containing P3 Sequences from Virulent Strains to Rsv1-Genotype Soybeans Is Mediated by Mutations in HC-Pro

In Rsv1-genotype soybean, Soybean mosaic virus (SMV)-N (an avirulent isolate of strain G2) elicits extreme resistance (ER) whereas strain SMV-G7 provokes a lethal systemic hypersensitive response (LSHR). SMV-G7d, an experimentally evolved variant of SMV-G7, induces systemic mosaic. Thus, for Rsv1-genotype soybean, SMV-N is avirulent whereas SMV-G7 and SMV-G7d are both virulent. Exploiting these differential interactions, we recently mapped the elicitor functions of SMV provoking Rsv1-mediated ER and LSHR to the N-terminal 271 amino acids of P3 from SMV-N and SMV-G7, respectively. The phenotype of both SMV-G7 and SMV-G7d were rendered avirulent on Rsv1-genotype soybean when the part of the genome encoding the N-terminus or the entire P3 cistron was replaced with that from SMV-N; however, reciprocal exchanges did not confer virulence to SMV-N-derived P3 chimeras. Here, we describe virulent SMV-N-derived P3 chimeras containing the full-length or the N-terminal P3 from SMV-G7 or SMV-G7d, with or without additional mutations in P3, that were selected on Rsv1-genotype soybean by sequential transfers on rsv1 and Rsv1-genotype soybean. Sequence analyses of the P3 and helper-component proteinase (HC-Pro) cistrons of progeny recovered from Rsv1-genotype soybean consistently revealed the presence of mutations in HC-Pro. Interestingly, the precise mutations in HC-Pro required for the adaptation varied among the chimeras. No mutation was detected in the HC-Pro of progeny passaged continuously in rsv1-genotype soybean, suggesting that selection is a consequence of pressure imposed by Rsv1. Mutations in HC-Pro alone failed to confer virulence to SMV-N; however, reconstruction of mutations in HC-Pro of the SMV-N-derived P3 chimeras resulted in virulence. Taken together, the data suggest that HC-Pro complementation of P3 is essential for SMV virulence on Rsv1-genotype soybean.

Pseudomonas syringae effector avrB confers soybean cultivar-specific avirulence on Soybean mosaic virus adapted for transgene expression but effector avrPto does not

Soybean mosaic virus (SMV) was adapted for transgene expression in soybean and used to examine the function of avirulence genes avrB and avrPto of Pseudomonas syringae pvs. glycinea and tomato, respectively. A cloning site was introduced between the P1 and HC-Pro genes in 35S-driven infectious cDNAs of strains SMV-N and SMV-G7. Insertion of the uidA gene or the green fluorescent protein gene into either modified cDNA and bombardment into primary leaves resulted in systemic expression that reflected the pattern of viral movement into uninoculated leaves. Insertion of avrB blocked symptom development and detectable viral movement in cv. Harosoy, which carries the Rpg1-b resistance gene corresponding to avrB, but not in cvs. Keburi or Hurrelbrink, which lack Rpg1-b. In Keburi and Hurrelbrink, symptoms caused by SMV carrying avrB appeared more quickly and were more severe than those caused by the virus without avrB. Insertion of avrPto enhanced symptoms in Harosoy, Hurrelbrink, and Keburi. This result was unexpected because avrPto was reported to confer avirulence on P. syringae pv. glycinea inoculated to Harosoy. We inoculated Harosoy with P syringae pv. glycinea expressing avrPto, but observed no hypersensitive reaction, avrPto-dependent induction of pathogenesis-related protein la, or limitation of bacterial population growth. In Hurrelbrink, avrPto enhanced bacterial multiplication and exacerbated symptoms. Our results establish SMV as an expression vector for soybean. They demonstrate that resistance triggered by avrB is effective against SMV, and that avrB and avrPto have general virulence effects in soybean. The results also led to a reevaluation of the reported avirulence activity of avrPto in this plant.

Experimental adaptation of an RNA virus mimics natural evolution.

ABSTRACT Identification of virulence determinants of viruses is of critical importance in virology. In search of such determinants, virologists traditionally utilize comparative genomics between a virulent and an avirulent virus strain and construct chimeras to map their locations. Subsequent comparison reveals sequence differences, and through analyses of site-directed mutants, key residues are identified. In the absence of a naturally occurring virulent strain, an avirulent strain can be functionally converted to a virulent variant via an experimental evolutionary approach. However, the concern remains whether experimentally evolved virulence determinants mimic those that have evolved naturally. To provide a direct comparison, we exploited a plant RNA virus, soybean mosaic virus (SMV), and its natural host, soybean. Through a serial in vivo passage experiment, the molecularly cloned genome of an avirulent SMV strain was converted to virulent variants on functionally immune soybean genotypes harboring resistance factor(s) from the complex Rsv1 locus. Several of the experimentally evolved virulence determinants were identical to those discovered through a comparative genomic approach with a naturally evolved virulent strain. Thus, our observations validate an experimental evolutionary approach to identify relevant virulence determinants of an RNA virus.

Recent Advances in In Planta Transient Expression and Silencing Systems for Soybean Using Viral Vectors

Transient methods for overexpressing and silencing plant genes enable rapid analysis of gene function and gene regulation. Several methods that avoid the traditional time- and labor-intensive generation of stable transgenic plants have been used recently in soybean and other plants to express or silence genes. In this post-genomic era, the sequences of the soybean genome and many other crop genomes are readily available. Genome sequences coupled with transient expression and silencing systems enable large-scale screens to associate genes with traits, or more detailed characterization of gene products and regulatory sequences. Recombinant plant viruses that can carry genetic payloads of whole genes or gene fragments provide convenient platforms as vectors for transient gene expression and silencing in soybean. This chapter focuses on seven viral vector systems that have been used in soybean for overexpression and/or virus-induced gene silencing (VIGS) applications. We discuss the features of the viral genomes, strategies for their use, applications in gene expression and/or VIGS, and future prospects to expand the utility of viral vectors for soybean improvement.