Functional divergence between soybean FLOWERING LOCUS T orthologues FT2a and FT5a in post-flowering stem growth.

Genes in the FLOWERING LOCUS T (FT) family integrate external and internal signals to control various aspects of plant development. In soybean (Glycine max), FT2a and FT5a play a major role in floral induction, but their roles in post-flowering reproductive development remain undetermined. Ectopic overexpression analyses revealed that FT2a and FT5a similarly induced flowering, but FT5a was markedly more effective than FT2a for the post-flowering termination of stem growth. The down-regulation of Dt1, a soybean orthologue of Arabidopsis TERMINAL FLOWER1, in shoot apices in early growing stages of FT5a-overexpressing plants was concomitant with highly up-regulated expression of APETALA1 orthologues. The Dt2 gene, a repressor of Dt1, was up-regulated similarly by the overexpression of FT2a and FT5a, suggesting that it was not involved in the control of stem termination by FT5a. In addition to the previously reported interaction with FDL19, a homologue of the Arabidopsis bZIP protein FD, both FT2a and FT5a interacted with FDL12, but only FT5a interacted with FDL06. Our results suggest that FT2a and FT5a have different functions in the control of post-flowering stem growth. A specific interaction of FT5a with FDL06 may play a key role in determining post-flowering stem growth in soybean.

Development of the VIGS System in the Dioecious Plant Silene latifolia.

(1) Background: Silene latifolia is a dioecious plant, whose sex is determined by XY-type sex chromosomes. Microbotryum lychnidis-dioicae is a smut fungus that infects S. latifolia plants and causes masculinization in female flowers, as if Microbotryum were acting as a sex-determining gene. Recent large-scale sequencing efforts have promised to provide candidate genes that are involved in the sex determination machinery in plants. These candidate genes are to be analyzed for functional characterization. A virus vector can be a tool for functional gene analyses; (2) Methods: To develop a viral vector system in S. latifolia plants, we selected Apple latent spherical virus (ALSV) as an appropriate virus vector that has a wide host range; (3) Results: Following the optimization of the ALSV inoculation method, S. latifolia plants were infected with ALSV at high rates in the upper leaves. In situ hybridization analysis revealed that ALSV can migrate into the flower meristems in S. latifolia plants. Successful VIGS (virus-induced gene silencing) in S. latifolia plants was demonstrated with knockdown of the phytoene desaturase gene. Finally, the developed method was applied to floral organ genes to evaluate its usability in flowers; (4) Conclusion: The developed system enables functional gene analyses in S. latifolia plants, which can unveil gene functions and networks of S. latifolia plants, such as the mechanisms of sex determination and fungal-induced masculinization.

Gentian (Gentiana triflora) prevents transmission of apple latent spherical virus (ALSV) vector to progeny seeds.

MAIN CONCLUSION: Gentian plants ( Gentiana triflora ) severely restrict apple latent spherical virus (ALSV) invasion to the gametes (pollens and ovules) and block seed transmission to progeny plants. Early flowering of horticultural plants can be induced by infection of ALSV vector expressing Flowering Locus T (FT) gene. In the present study, flowering of gentian plants was induced by infection with an ALSV vector expressing a gentian FT gene and the patterns of seed transmission of ALSV in gentian were compared with those in apple and Nicotiana benthamiana. Infection of gentian progeny plants with ALSV was examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and enzyme-linked immunosorbent assay (ELISA). ALSV was not transmitted to the progeny gentian plants, whereas small proportions of apple and N. benthamiana progeny plants are infected with ALSV. The in situ hybridization analyses indicated that ALSVs are not present in gentian pollen and ovules, but detected in most of gametes in apple and N. benthamiana. Collectively, these results suggest that seed transmission of ALSV is blocked in gentian plants through the unknown barriers present in their gametes. On the other hand, apple and N. benthamiana seem to minimize ALSV seed transmission by inhibiting viral propagation in embryos.

Allelic variants of the esterase gene W14/15 differentially regulate overwinter survival in perennial gentian (Gentiana L.).

Overwinter survival has to be under critical regulation in the lifecycle of herbaceous perennial plants. Gentians (Gentiana L.) maintain their perennial life style through producing dormant and freezing-tolerant overwinter buds (OWBs) to overcome cold winter. However, the mechanism acting on such an overwinter survival and the genes/proteins contributing to it have been poorly understood. Previously, we identified an OWB-enriched protein W14/15, a member of a group of α/ß hydrolase fold superfamily that is implicated in regulation of hormonal action in plants. The W14/15 gene has more than ten variant types in Gentiana species. However, roles of the W14/15 gene in OWB survival and functional difference among those variants have been unclear. In the present study, we examined whether the W14/15 gene variants are involved in the mechanism acting on overwinter survival, by crossing experiments using cultivars carrying different W14/15 variant alleles and virus-induced gene silencing experiments. We found that particular types of the W14/15 variants (W15a types) contributed toward obtaining high ability of overwinter survival, while other types (W14b types) did not, or even interfered with the former type gene. This study demonstrates two findings; first, contribution of esterase genes to winter hardiness, and second, paired set or paired partner among the allelic variants determines the ability of overwinter survival.

Apple latent spherical virus vector-induced flowering for shortening the juvenile phase in Japanese gentian and lisianthus plants.

MAIN CONCLUSION: Infection by apple latent spherical virus (ALSV) vectors that promote the expression of Arabidopsis thaliana FLOWERING LOCUS T ( AtFT ) or Gentiana triflora GtFT s accelerates flowering in gentian and lisianthus plants. Apple latent spherical virus (ALSV) has isometric virus particles (25 nm in diameter) that contain two ssRNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp20, and Vp24). ALSV vectors are used for foreign gene expression and virus-induced gene silencing in a broad range of plant species. Here, we report the infection by ALSV vectors that express FLOWERING LOCUS T (AtFT) from Arabidopsis thaliana or its homolog GtFT1 from Gentiana triflora in three gentian cultivars ('Iwate Yume Aoi' [early flowering], 'Iwate' [medium flowering], and 'Alta' [late flowering]), and two lisianthus cultivars ('Newlination Pink ver. 2' and 'Torukogikyou daburu mikkusu') promotes flowering within 90 days post-inoculation using particle bombardment. Additionally, seedlings from the progeny of the early-flowering plants were tested by tissue blot hybridization, and the results showed that ALSV was not transmitted to the next generation. The promotion of flowering in the family Gentianaceae by ALSV vectors shortened the juvenile phase from 1-3 years to 3-5 months, and thus, it could be considered as a new plant breeding technique in ornamental gentian and lisianthus plants.

The Soybean-Specific Maturity Gene E1 Family of Floral Repressors Controls Night-Break Responses through Down-Regulation of FLOWERING LOCUS T Orthologs.

Photoperiodism is a rhythmic change of sensitivity to light, which helps plants to adjust flowering time according to seasonal changes in daylength and to adapt to growing conditions at various latitudes. To reveal the molecular basis of photoperiodism in soybean (Glycine max), a facultative short-day plant, we analyzed the transcriptional profiles of the maturity gene E1 family and two FLOWERING LOCUS T (FT) orthologs (FT2a and FT5a). E1, a repressor for FT2a and FT5a, and its two homologs, E1-like-a (E1La) and E1Lb, exhibited two peaks of expression in long days. Using two different approaches (experiments with transition between light and dark phases and night-break experiments), we revealed that the E1 family genes were expressed only during light periods and that their induction after dawn in long days required a period of light before dusk the previous day. In the cultivar Toyomusume, which lacks the E1 gene, virus-induced silencing of E1La and E1Lb up-regulated the expression of FT2a and FT5a and led to early flowering. Therefore, E1, E1La, and E1Lb function similarly in flowering. Regulation of E1 and E1L expression by light was under the control of E3 and E4, which encode phytochrome A proteins. Our data suggest that phytochrome A-mediated transcriptional induction of E1 and its homologs by light plays a critical role in photoperiodic induction of flowering in soybean.

Isolation and characterization of the C-class MADS-box gene involved in the formation of double flowers in Japanese gentian.

BACKGROUND: Generally, double-flowered varieties are more attractive than single-flowered varieties in ornamental plants. Japanese gentian is one of the most popular floricultural plants in Japan, and it is desirable to breed elite double-flowered cultivars. In this study, we attempted to characterize a doubled-flower mutant of Japanese gentian. To identify the gene that causes the double-flowered phenotype in Japanese gentian, we isolated and characterized MADS-box genes. RESULTS: Fourteen MADS-box genes were isolated, and two of them were C-class MADS-box genes (GsAG1 and GsAG2). Both GsAG1 and GsAG2 were categorized into the PLE/SHP subgroup, rather than the AG/FAR subgroup. In expression analyses, GsAG1 transcripts were detected in the second to fourth floral whorls, while GsAG2 transcripts were detected in only the inner two whorls. Transgenic Arabidopsis expressing GsAG1 lacked petals and formed carpeloid organs instead of sepals. Compared with a single-flowered gentian cultivar, a double-flowered gentian mutant showed decreased expression of GsAG1 but unchanged expression of GsAG2. An analysis of the genomic structure of GsAG1 revealed that the gene had nine exons and eight introns, and that a 5,150-bp additional sequence was inserted into the sixth intron of GsAG1 in the double-flowered mutant. This insert had typical features of a Ty3/gypsy-type LTR-retrotransposon, and was designated as Tgs1. Virus-induced gene silencing of GsAG1 by the Apple latent spherical virus vector resulted in the conversion of the stamen to petaloid organs in early flowering transgenic gentian plants expressing an Arabidopsis FT gene. CONCLUSIONS: These results revealed that GsAG1 plays a key role as a C-functional gene in stamen organ identity. The identification of the gene responsible for the double-flowered phenotype will be useful in further research on the floral morphogenesis of Japanese gentian.

Reduced generation time of apple seedlings to within a year by means of a plant virus vector: a new plant-breeding technique with no transmission of genetic modification to the next generation.

Fruit trees have a long juvenile phase. For example, the juvenile phase of apple (Malus × domestica) generally lasts for 5-12 years and is a serious constraint for genetic analysis and for creating new apple cultivars through cross-breeding. If modification of the genes involved in the transition from the juvenile phase to the adult phase can enable apple to complete its life cycle within 1 year, as seen in herbaceous plants, a significant enhancement in apple breeding will be realized. Here, we report a novel technology that simultaneously promotes expression of Arabidopsis FLOWERING LOCUS T gene (AtFT) and silencing of apple TERMINAL FLOWER 1 gene (MdTFL1-1) using an Apple latent spherical virus (ALSV) vector (ALSV-AtFT/MdTFL1) to accelerate flowering time and life cycle in apple seedlings. When apple cotyledons were inoculated with ALSV-AtFT/MdTFL1 immediately after germination, more than 90% of infected seedlings started flowering within 1.5-3 months, and almost all early-flowering seedlings continuously produced flower buds on the lateral and axillary shoots. Cross-pollination between early-flowering apple plants produced fruits with seeds, indicating that ALSV-AtFT/MdTFL1 inoculation successfully reduced the time required for completion of the apple life cycle to 1 year or less. Apple latent spherical virus was not transmitted via seeds to successive progenies in most cases, and thus, this method will serve as a new breeding technique that does not pass genetic modification to the next generation.

Distinct associations between gratitude, self-esteem, and optimism with subjective and psychological well-being among Japanese individuals.

BACKGROUND: Mounting evidence suggests that the effectiveness of positive psychology interventions is influenced by a variety of factors, including cultural context. Identifying intervention targets that can effectively contribute to improving individual well-being under these boundary conditions is a crucial step when developing viable interventions. To this end, we examined how gratitude disposition, self-esteem, and optimism relate to the subjective well-being (SWB) and psychological well-being (PWB) of Japanese individuals. METHODS: Multivariate regression analysis was employed to quantify the unique relationships between the three potential intervention targets and both SWB and PWB, while accounting for the influence of other variables. Participants (N = 71) also engaged in a 4-week experience sampling study to explore how gratitude, self-esteem and optimism shape the link between momentary affective states in everyday life and evaluations of day satisfaction. RESULTS: Multivariate regression analysis revealed that self-esteem was predominantly more strongly associated with SWB compared to gratitude disposition, whereas gratitude disposition was more strongly associated with the PWB dimensions, particularly personal growth, positive relations with others and purpose in life. Experience sampling data indicated that while both gratitude disposition and self-esteem moderated the association between momentary positive affect and day satisfaction evaluations, they did so in opposite ways; greater gratitude disposition strengthened the association, while greater self-esteem weakened it. CONCLUSIONS: Overall, the current results suggest that while gratitude, self-esteem, and optimism influence individual well-being as a whole, they likely play distinct roles in facilitating SWB and PWB in the studied cohort.

Duration of mood effects following a Japanese version of the mood induction task.

Researchers have employed a variety of methodologies to induce positive and negative mood states in study participants to investigate the influence that mood has on psychological, physiological, and cognitive processes both in health and illness. Here, we investigated the effectiveness and the duration of mood effects following the mood induction task (MIT), a protocol that combines mood-inducing sentences, auditory stimuli, and autobiographical memory recall in a cohort of healthy Japanese adult individuals. In Study 1, we translated and augmented the mood-inducing sentences originally proposed by Velten in 1968 and verified that people perceived the translations as being largely congruent with the valence of the original sentences. In Study 2, we developed a Japanese version of the mood induction task (J-MIT) and examined its effectiveness using an online implementation. Results based on data collected immediately after induction showed that the J-MIT was able to modulate the mood in the intended direction. However, mood effects were not observed during the subsequent performance of a cognitive task, the Tower of London task, suggesting that the effects did not persist long enough. Overall, the current results show that mood induction procedures such as the J-MIT can alter the mood of study participants in the short term; however, at the same time, they highlight the need to further examine how mood effects evolve and persist through time to better understand how mood induction protocols can be used to study affective processes more effectively.

A MYB transcription factor controls flower color in soybean.

Purple-blue flower of soybean (Glycine max [L.] Merr.) is controlled by the W2 locus. Previous studies revealed that a MYB transcription factor gene GmMYB-G20-1 was located at a position similar to the W2 gene and that a base substitution generated a stop codon in the MYB domains of 2 soybean lines with purple-blue flowers. This study was conducted to confirm the relationship between GmMYB-G20-1 and the W2 gene. Cleaved amplified polymorphic sequence analysis to detect the base substitution suggested that a similar mutation occurred in 2 other soybean lines having purple-blue flowers, 037-E-8, and Yogetsu 1-blue. Thus, all genotypes having purple-blue flowers had identical base substitutions. To verify the function of GmMYB-G20-1, apple latent spherical virus (ALSV) vectors were constructed to perform virus-induced gene silencing of GmMYB-G20-1. A cultivar Harosoy with purple flowers (W2W2) was infected by the empty ALSV vector (wtALSV) or the GmMYB-G20-1-ALSV vector containing a fragment (nucleotide position 685-885) of GmMYB-G20-1. Plants infected by empty vectors had only purple flowers. In contrast, most flowers of plants infected with GmMYB-G20-1-ALSV had irregular gray/blue sectors in flower petals and some of the flowers had almost gray/blue petals. These results strongly suggest that silencing of GmMYB-G20-1 can alter flower color and that it may correspond to the W2 gene.

Combining sparseness and smoothness improves classification accuracy and interpretability.

Sparse logistic regression (SLR) has been shown to be a useful method for decoding high-dimensional fMRI and MEG data by automatically selecting relevant feature dimensions. However, when applied to signals with high spatio-temporal correlations, SLR often over-prunes the feature space, which can result in overfitting and weight vectors that are difficult to interpret. To overcome this problem, we investigate a modification of ℓ1-normed sparse logistic regression, called smooth sparse logistic regression (SSLR), which has a spatio-temporal "smoothing" prior that encourages weights that are close in time and space to have similar values. This causes the classifier to select spatio-temporally continuous groups of features, whereas SLR classifiers often select a scattered collection of independent features. We applied the method to both simulation data and real MEG data. We found that SSLR consistently increases classification accuracy, and produces weight vectors that are more meaningful from a neuroscientific perspective.

Efficient virus-induced gene silencing system in pumpkin (Cucurbita maxima) using apple latent spherical virus vector.

Crude-sap of apple latent spherical virus (ALSV)-infected Chenopodium quinoa leaves was rub-inoculated on the expanded cotyledons of various Cucurbitaceae plants. Most of the species were systemically infected with the virus without obvious symptoms, except pumpkin (Cucurbita maxima). In pumpkin, the ALSV infection was restricted to inoculated cotyledons; it did not spread to the upper true leaves. In situ hybridization showed that the ALSV was confined to part of the cotyledon tissues and it did not invade the phloem tissue, when inoculated at the expanded cotyledon stage. However, when total RNAs from ALSV-infected C. quinoa leaves were inoculated into the cotyledons immediately after germination (folded cotyledon stage) using particle bombardment, ALSV efficiently caused systemic infection. Systemic infection of pumpkin seedlings occurred only when the cotyledons were inoculated within a few days after germination. No systemic infection was observed in the seedlings 4 days after germination. In the grafting test, ALSV was not transmitted from the infected rootstocks to the healthy scions of pumpkins. An efficient virus-induced gene silencing system for pumpkins was established, in which infection with ALSV vectors harboring the phytoene desaturase or sulfur gene fragments resulted in a uniform phenotype in the true leaves of pumpkin seedlings.

Promotion of flowering and reduction of a generation time in apple seedlings by ectopical expression of the Arabidopsis thaliana FT gene using the Apple latent spherical virus vector.

Tree crops have a long juvenile period which is a serious constraint for genetic improvement and experimental research. For example, apple remains in a juvenile phase for more than five years after seed germination. Here, we report about induction of rapid flowering in apple seedlings using the Apple latent spherical virus (ALSV) vector expressing a FLOWERING LOCUS T (FT) gene from Arabidopsis thaliana. Apple seedlings could be flowered at 1.5-2 months after inoculation to cotyledons of seeds just after germination with ALSV expressing the FT gene. A half of precocious flowers was normal in appearance with sepals, petals, stamens, and pistils. Pollen from a precocious flower successfully pollinated flowers of 'Fuji' apple from which fruits developed normally and next-generation seeds were produced. Our system using the ALSV vector promoted flowering time of apple seedlings within two months after germination and shortened the generation time from seed germination to next-generation seed maturation to within 7 months when pollen from precocious flowers was used for pollination.

Expression of FLOWERING LOCUS T from Arabidopsis thaliana induces precocious flowering in soybean irrespective of maturity group and stem growth habit.

The flowering integrator gene FLOWERING LOCUS T (FT) in Arabidopsis thaliana is conserved between diverse plant species and is thought to be the flowering signal ''florigen'', a universal long-distance mobile signal. In soybean, two FT homologs having a function to induce flowering in Arabidopsis have been identified. In this study, we showed that the expression of FT from Arabidopsis by the Apple latent spherical virus (ALSV) vector promoted precocious flowering and terminated vegetative growth in a wide range of genotypes of soybean, without using a short-day treatment. Four determinate and two indeterminate cultivars, infected with ALSV expressing FT (FT-ALSV), set flower buds on shoot apices and terminated vegetative growth at the fourth- to seventh-node stages under long-day conditions. In contrast, non-infected, healthy plants did not set flower buds on shoot apices at the same stage under the same conditions. After flowering, soybean cultivars infected with FT-ALSV, belonging to different maturity groups and stem growth habits, matured and produced seeds. The results suggest that the basic flowering pathway controlled by FT in A. thaliana might also be conserved in soybean. A system for precocious flowering and shortening of generation time using FT-ALSV would be a useful and novel technology for efficient soybean breeding.

Enhanced academic motivation in university students following a 2-week online gratitude journal intervention.

BACKGROUND: Past studies have associated gratitude interventions with a host of positive outcomes. However, there is a dearth of research regarding the impact such interventions have on the academic motivation of university students, thought to be a primary determinant of academic achievement and overall satisfaction with school activities. Here, we examined the effects of a 2-week online gratitude journal intervention on the academic motivation of university students. METHODS: Eighty-four students were randomly assigned to either an active manipulation group (gratitude group) or a neutral control group. In the first 6 days of each week, participants in the gratitude group were asked to log in to the online system once a day and list up to five things they had felt grateful for. They were also requested to evaluate various aspects of their daily lives. Participants in the control group were only requested to perform the daily self-evaluations. Academic motivation was assessed using the Academic Motivation Scale (AMS), which conceptualizes motivation in academic settings as being composed by three different components, i.e., intrinsic motivation, extrinsic motivation and amotivation, the latter being associated with the perceived lack of contingency between actions and outcomes. Responses were collected 5 times: before group assignment (baseline), 1 week after the start of the intervention, immediately after the intervention, and at two follow-ups, 1 and 3 months after the intervention. RESULTS: Analysis using a self-determination index derived from the AMS components showed that participants who regularly engaged with the gratitude journal task displayed significant enhancements in academic motivation. Additional analysis revealed that the enhancements were driven by decreases in the levels of amotivation. Furthermore, follow-up data showed that there were no signs that such enhancements had receded 3 months after the end of the intervention. Improvements in academic motivation were not observed among participants in the control group. CONCLUSIONS: The current results provide evidence that gratitude interventions can positively impact the academic motivation of university students. More broadly, they show that the effects extend well beyond the realm of typically assessed measures of individual well-being, and can effectively regulate a fundamental component of goal-directed behavior such as motivation.

Assessing changes in mood state in university students following short-term study abroad.

Short-term study-abroad (STSA) programs provide a more accessible alternative for students who would otherwise not consider engaging in academic activities overseas. Though improvements in the levels of intercultural sensitivity and general academic aspects attained by STSA programs have been previously examined, much less is known regarding the impact such programs have in the mood of students. Here, we examined changes in mood state associated with participation in an STSA program in a group of Japanese university students. Mood states were assessed using the Profile of Mood States (POMS), the Satisfaction With Life Scale (SWLS), and the Gratitude Questionnaire (GQ-6). Results indicated that the POMS mean scores of Vigor-Activity and SWLS peaked at the time immediately following participation in the STSA program; moreover, the same scores were found to be at comparable levels even one month after the end of the program. These results indicate that participation in STSA programs can positively influence the mood state of university students, suggesting that the benefits associated with participation in such programs extend beyond typically reported improvements in the academic domain.

The observant mind: self-awareness of attentional status.

Visual perception is dependent not only on low-level sensory input but also on high-level cognitive factors such as attention. In this paper, we sought to determine whether attentional processes can be internally monitored for the purpose of enhancing behavioural performance. To do so, we developed a novel paradigm involving an orientation discrimination task in which observers had the freedom to delay target presentation--by any amount required--until they judged their attentional focus to be complete. Our results show that discrimination performance is significantly improved when individuals self-monitor their level of visual attention and respond only when they perceive it to be maximal. Although target delay times varied widely from trial-to-trial (range 860 ms-12.84 s), we show that their distribution is Gaussian when plotted on a reciprocal latency scale. We further show that the neural basis of the delay times for judging attentional status is well explained by a linear rise-to-threshold model. We conclude that attentional mechanisms can be self-monitored for the purpose of enhancing human decision-making processes, and that the neural basis of such processes can be understood in terms of a simple, yet broadly applicable, linear rise-to-threshold model.

[Virus-induced gene silencing as a tool for analysis of gene functions in plants].

Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antivirus defense mechanism in plants and has been shown to have great potential in plant reverse genetics. When the virus vector carries sequences of plant genes, virus infection triggers VIGS that results in the degradation of endogenous mRNAs homologous to the plant genes. The system is well established in Nicotiana benthamiana and several reliable VIGS vectors have been developed for other plant species including important agricultural crops. Here, we describe the use of VIGS technology to determine gene function and plant virus vectors for induction of VIGS in plants.

Apple Russet Ring and Apple Green Crinkle Diseases: Fulfillment of Koch's Postulates by Virome Analysis, Amplification of Full-Length cDNA of Viral Genomes, in vitro Transcription of Infectious Viral RNAs, and Reproduction of Symptoms on Fruits of Apple Trees Inoculated With Viral RNAs.

Apple russet ring and apple green crinkle are graft-transmitted diseases first reported more than 60 years ago, but at present, no association between a specific virus (variant) and the disease has been clearly demonstrated. In this study, we conducted the following series of experiments to identify the causal viruses (variants) of these apple diseases; (1) comprehensive analysis by next-generation sequencing of all viruses in each apple tree affected with russet ring or green crinkle disease, (2) amplification of full-length genomic cDNA of viruses using primers containing the T3 promoter and the in vitro transcription of infectious viral RNAs, (3) inoculation of viral RNA transcripts to both herbaceous and apple plants, (4) analysis of sequence variants of viruses present in infected plants, (5) back-inoculation of sequence variants of candidate viruses to apple seedlings combined with the virus-induced flowering technology using the apple latent spherical virus vector to reproduce the symptom on the fruit as soon as possible, and (6) reproduction of symptoms on the fruits of apple trees inoculated with sequence variants and the re-isolation of each virus variant from apples showing fruit symptoms. The results showed that one of the sequence variants of the apple chlorotic leaf spot virus causes a characteristic ring-shaped rust on the fruits of infected apple trees and that a sequence variant of the apple stem pitting virus probably causes green crinkle symptoms on an infected apple fruit. Thus, we were able to fulfill Koch's postulates to prove the viral etiology of both the apple russet ring and green crinkle diseases. We also propose an experimental system that can prove whether a virus found in diseased tissues is the pathogen responsible for the diseases when the etiology is undetermined.