Kim D. Bowman

Transcriptome Profiling of Citrus Fruit Response to Huanglongbing Disease

Huanglongbing (HLB) or “citrus greening” is the most destructive citrus disease worldwide. In this work, we studied host responses of citrus to infection with Candidatus Liberibacter asiaticus (CaLas) using next-generation sequencing technologies. A deep mRNA profile was obtained from peel of healthy and HLB-affected fruit. It was followed by pathway and protein-protein network analysis and quantitative real time PCR analysis of highly regulated genes. We identified differentially regulated pathways and constructed networks that provide a deep insight into the metabolism of affected fruit. Data mining revealed that HLB enhanced transcription of genes involved in the light reactions of photosynthesis and in ATP synthesis. Activation of protein degradation and misfolding processes were observed at the transcriptomic level. Transcripts for heat shock proteins were down-regulated at all disease stages, resulting in further protein misfolding. HLB strongly affected pathways involved in source-sink communication, including sucrose and starch metabolism and hormone synthesis and signaling. Transcription of several genes involved in the synthesis and signal transduction of cytokinins and gibberellins was repressed while that of genes involved in ethylene pathways was induced. CaLas infection triggered a response via both the salicylic acid and jasmonic acid pathways and increased the transcript abundance of several members of the WRKY family of transcription factors. Findings focused on the fruit provide valuable insight to understanding the mechanisms of the HLB-induced fruit disorder and eventually developing methods based on small molecule applications to mitigate its devastating effects on fruit production.

Gene regulatory networks elucidating huanglongbing disease mechanisms.

Next-generation sequencing was exploited to gain deeper insight into the response to infection by Candidatus liberibacter asiaticus (CaLas), especially the immune disregulation and metabolic dysfunction caused by source-sink disruption. Previous fruit transcriptome data were compared with additional RNA-Seq data in three tissues: immature fruit, and young and mature leaves. Four categories of orchard trees were studied: symptomatic, asymptomatic, apparently healthy, and healthy. Principal component analysis found distinct expression patterns between immature and mature fruits and leaf samples for all four categories of trees. A predicted protein – protein interaction network identified HLB-regulated genes for sugar transporters playing key roles in the overall plant responses. Gene set and pathway enrichment analyses highlight the role of sucrose and starch metabolism in disease symptom development in all tissues. HLB-regulated genes (glucose-phosphate-transporter, invertase, starch-related genes) would likely determine the source-sink relationship disruption. In infected leaves, transcriptomic changes were observed for light reactions genes (downregulation), sucrose metabolism (upregulation), and starch biosynthesis (upregulation). In parallel, symptomatic fruits over-expressed genes involved in photosynthesis, sucrose and raffinose metabolism, and downregulated starch biosynthesis. We visualized gene networks between tissues inducing a source-sink shift. CaLas alters the hormone crosstalk, resulting in weak and ineffective tissue-specific plant immune responses necessary for bacterial clearance. Accordingly, expression of WRKYs (including WRKY70) was higher in fruits than in leaves. Systemic acquired responses were inadequately activated in young leaves, generally considered the sites where most new infections occur.

Overexpression of a citrus NDR1 ortholog increases disease resistance in Arabidopsis

Emerging devastating diseases, such as Huanglongbing (HLB) and citrus canker, have caused tremendous losses to the citrus industry worldwide. Genetic engineering is a powerful approach that could allow us to increase citrus resistance against these diseases. The key to the success of this approach relies on a thorough understanding of defense mechanisms of citrus. Studies of Arabidopsis and other plants have provided a framework for us to better understand defense mechanisms of citrus. Salicylic acid (SA) is a key signaling molecule involved in basal defense and resistance (R) gene-mediated defense against broad-spectrum pathogens. The Arabidopsis gene NDR1 (NON-RACE-SPECIFIC DISEASE RESISTANCE 1) is a positive regulator of SA accumulation and is specifically required for signaling mediated by a subset of R genes upon recognition of their cognate pathogen effectors. Our bioinformatic analysis identified an ortholog of NDR1 from citrus, CsNDR1. Overexpression of CsNDR1 complemented susceptibility conferred by the Arabidopsis ndr1-1 mutant to Pseudomonas syringae strains and also led to enhanced resistance to an oomycete pathogen Hyaloperonospora arabidopsidis. Such heightened resistance is associated with increased SA production and expression of the defense marker gene PATHOGENESIS RELATED 1 (PR1). In addition, we found that expression of PR1 and accumulation of SA were induced to modest levels in citrus infected with Candidatus Liberibacter asiaticus, the bacterial pathogen associated with HLB disease. Thus, our data suggest that CsNDR1 is a functional ortholog of Arabidopsis NDR1. Since Ca. L. asiaticus infection only activates modest levels of defense responses in citrus, we propose that genetically increasing SA/NDR1-mediated pathways could potentially lead to enhanced resistance against HLB, citrus canker, and other destructive diseases challenging global citrus production.

GUS expression driven by constitutive and phloem-specific promoters in citrus hybrid US-802

Transgenic solutions are being widely explored to develop huanglongbing (HLB) resistance in citrus. A critical component of a transgenic construct is the promoter, which determines tissue specificity and level of target gene expression. This study compares the characteristics of five promoters regulating the beta-glucuronidase (GUS) reporter gene in the trifoliate hybrid rootstock US-802. Two of the selected promoters direct high levels of constitutive transgene expression in other dicotyledonous plants: 2X35S, the tandem-repeat promoter of the cauliflower mosaic virus 35S gene and bul409S, a truncation of the potato polyubiquitin promoter. Because Candidatus Liberibacter, the Gram-negative bacterium associated with HLB, infects only the phloem tissue, it may be advantageous to limit transgene expression to the vascular tissue and reduce expression in the fruit. Thus, we also tested three promoters that demonstrate phloem specificity when transformed and expressed in other plants: WDV, from wheat dwarf geminivirus; AtSUC2, the sucrose-H+ symporter gene promoter from Arabidopsis; and CsSUS, the sucrose synthase promoter from citrus. Histochemical staining for GUS activity was observed throughout leaf and stem tissues for the constitutive promoters, while the three phloem-specific promoters largely showed the expected tissue-specific staining. Expression of GUS in some individual transformants with promoters CsSUS and WDV appeared leaky, with some laminar tissue staining. Relative quantification of qRT-PCR data revealed a wide range of mRNA abundance from transgenics with each of the five promoters. Fluorometry also revealed that GUS activity differed depending on the promoter used, but mRNA levels and enzyme activity were not highly correlated.

A Dark Incubation Period Is Important for Agrobacterium-Mediated Transformation of Mature Internode Explants of Sweet Orange, Grapefruit, Citron, and a Citrange Rootstock

Background Citrus has an extended juvenile phase and trees can take 2–20 years to transition to the adult reproductive phase and produce fruit. For citrus variety development this substantially prolongs the time before adult traits, such as fruit yield and quality, can be evaluated. Methods to transform tissue from mature citrus trees would shorten the evaluation period via the direct production of adult phase transgenic citrus trees. Methodology/Principal Findings Factors important for promoting shoot regeneration from internode explants from adult phase citrus trees were identified and included a dark incubation period and the use of the cytokinin zeatin riboside. Transgenic trees were produced from four citrus types including sweet orange, citron, grapefruit, and a trifoliate hybrid using the identified factors and factor settings. Significance The critical importance of a dark incubation period for shoot regeneration was established. These results confirm previous reports on the feasibility of transforming mature tissue from sweet orange and are the first to document the transformation of mature tissue from grapefruit, citron, and a trifoliate hybrid.

Transmission Rates of ‘ Ca. Liberibacter asiaticus’ by Asian Citrus Psyllid Are Enhanced by the Presence and Developmental Stage of Citrus Flush

Abstract Asian citrus psyllid (Diaphorina citri Kuwayama) transmits a bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) putatively responsible for a devastating citrus disease known as Asiatic huanglongbing (HLB) (citrus greening disease). The psyllid and disease have invaded many citrus-growing regions including the United States, where the disease is seriously jeopardizing the Florida citrus industry. We recently concluded research that showed CLas transmission rates are increased when citrus flush is present. Flush is any new leaf growth ranging in development from first emergence up until the leaves are fully expanded yet still tender. In an experiment with seedlings of a rootstock cultivar ‘US-942’, a 1-wk infestation of 20 Asian citrus psyllids from an infected colony resulted in 53–60% of seedlings becoming infected when flush was present compared with only 7% when no flush was present. In a second experiment with ‘US-942’, 77–97% of seedlings became infected when flush was present compared with 40% when no flush was present. A similar experiment with ‘Valencia’ sweet orange resulted in 23, 80, and 3% seedlings becoming infected when young, older, or no flush was present, respectively. Young plants are therefore more likely to contract HLB if flush is present, with older flush promoting higher infection rates under the conditions of this study. Based on this finding, healthy citrus should be protected from Asian citrus psyllid infestations throughout a flush. To evaluate germplasm for CLas resistance, inoculations using infected Asian citrus psyllid would best be achieved if flush is present.

Rootstock Effects on Fruit Quality among ‘Ray Ruby’ Grapefruit Trees Grown in the Indian River District of Florida

The objective of this experiment was to compare fruit-quality parameters of ‘Ray Ruby’ grapefruit grown on seven rootstocks. Four recent releases from the United States Department of Agriculture (USDA) rootstock breeding program, ‘US-852’, ‘US897’, ‘US-942’, and ‘US-812’ (all Citrus reticulata 3 Poncirus trifoliata hybrids), ‘x639’ (C. reticulata3 P. trifoliata), along with industry-standard ‘Sour Orange’ and ‘Swingle’ citrumelo were evaluated in a commercial orchard trial in Indian River County, FL. Fruit-quality data were collected in 2011–12 (eight harvests), 2012–13 (five harvests), and 2014 (single harvest). In each season, rootstock effects on fruit size, total solids, and solids acid ratio were significant. ‘Sour orange’ and ‘Swingle’ produced the largest fruit, whereas ‘US-897’ (a semidwarfing rootstock) produced the smallest fruit. Peel thickness (measured only in the 2011–12 season) was greatest in ‘Sour Orange’ early in the season, but not toward the end of the season. Misshapen (‘‘sheep-nosed’’) fruit occurred more frequently on ‘Sour Orange’ than on other rootstocks, although the incidence of sheepnosing was minor. Analysis of variance (ANOVA) for fruit-quality data collected in January of each of the 3 years confirmed that ‘Sour Orange’ and ‘Swingle’ produced the largest fruit and ‘US-897’ produced the smallest fruit. Total solids were the highest in ‘US-897’ and the lowest in ‘x639’ and ‘US-852’. Taken together, our data indicate that ‘US-942’ and ‘US-897’ rootstocks produced fruit with quality characteristics that equaled or exceeded ‘Sour Orange’ and ‘Swingle’, the two most common rootstocks used in the Indian River district. The Indian River district of Florida is the world’s major production region for grapefruit (Citrus paradisi Macf.), and Indian River grapefruit are valued for their high quality. In contrast to the deep, well-drained sandy soil characteristic of Florida’s ‘‘central ridge,’’ soils in the Indian River district are typically shallow, poorly drained, and referred to as ‘‘flatwoods’’ (Harris et al., 2010). Grapefruit, like all commercial citrus, is produced as a composite tree consisting of a scion grafted onto a rootstock. Historically, ‘Sour orange’ (Citrus aurantium L.), believed to be a hybrid of Citrus maxima · Citrus reticulata Blanco (Grosser et al., 2004), has been a favored rootstock for citrus in the Indian River district. However, with the exception of lemons, citrus scions grown on ‘Sour orange’ are susceptible to Citrus tristeza virus (CTV), and when the brown citrus aphid, vector of CTV, arrived in Florida, ‘Sour orange’ fell out of favor. Subsequently, ‘Swingle’ citrumelo [‘Duncan’ grapefruit (Citrus paradisi) · Poncirus trifoliata (L.) Raf.] replaced ‘Sour orange’ as the most commonly used rootstock for citrus in the Indian River district (Stover and Castle, 2002). Unfortunately, although ‘Swingle’ is well suited as a rootstock in the deep sands of the Florida central ridge, it has proven to be a poor rootstock for the flatwoods soils typical of the Indian River district (Bauer et al., 2005; Castle et al., 2016). Improved citrus rootstocks for grapefruit are essential if sustainable production is to be maintained (Castle et al., 2011). Citrus rootstock breeding has been conducted by the USDA in Florida for more than a century (Soost and Rouse, 1996). The use of hybrids between Citrus spp. and Poncirus trifoliata, a member of the Rutaceae that is sexually compatible with Citrus, has been, by far, the most productive strategy for the development of new citrus rootstocks in the USDA breeding program and in other citrus rootstock breeding programs. Between 1999 and 2010, five new citrus rootstock cultivars were released by the USDA. Four of these new rootstocks (‘US-852’, ‘US-812’, ‘US-897’, and ‘US-942’) are hybrids of Citrus reticulata (mandarin) · Poncirus trifoliata (trifoliate orange). ‘US-852’, released in 1999 (Bowman and Wutscher, 1999), is a hybrid of ‘Changsha’ mandarin · ‘English Large Flowered’ trifoliate orange. ‘US-812’, released in 2001 (Bowman, 2001; Bowman and Rouse, 2006), is a hybrid of ‘Sunki’ mandarin and ‘Benecke’ trifoliate orange. Trees grown on ‘US-812’ rootstock are medium-sized with a wide range of scions and consistently yield large quantities of good-quality fruit. ‘US-812’ also exhibits resistance to citrus blight and has the CTV resistance gene ctvR. ‘US-897’ was released in 2007 (Bowman, 2007) and is a hybrid of ‘Cleopatra’ mandarin · ‘Flying Dragon’ trifoliate orange. This hybrid rootstock has field tolerance to the Phytophthora–Diaprepes disease complex, resistance to CTV, good fruit productivity, and good fruit quality (Bowman et al., 2016a). Trees produced on ‘US-897’ are compact, making this rootstock attractive to growers who want to increase tree density. This is especially important in Florida as tree densities are increasing from a previous average of 140 trees per acre to as many as 300 trees per acre currently. ‘US-942’ citrus rootstock was released in 2010 (Bowman and McCollum, 2010). Trees grown on ‘US-942’ are medium-sized with a wide range of scions and consistently yield large quantities of good-quality fruit (Bowman et al., 2016a). ‘US-942’ also exhibits field tolerance to the Phytophthora–Diaprepes disease complex, resistance to citrus blight, has the CTV resistance gene ctvR, and is a hybrid of ‘Sunki’ mandarin · ‘Flying Dragon’ trifoliate orange. The ‘x639’ rootstock, developed in South Africa in the early 1950s, is a hybrid of ‘Cleopatra’ mandarin (Citrus reticulata) · Table 1. Trial planting details and proportion of trees dead in 2011 (5–6 yr age). Rootstock Planting arrangement Number of trees planted Number of trees died Percent tree death US-897 6 · 112 672 12 1.8 b US-812 6 · 76 456 12 2.6 b US-852 6 · 76 456 14 3.1 b Swingle 6 · 76 456 16 3.5 b US-942 6 · 112 672 24 3.6 b x639 6 · 76 456 23 5.0 b Sour orange 6 · 76 456 48 10.5 a Mean separations for significant analysis of variance within columns, by Duncan’s multiple range test at P < 0.05. Planting arrangement = reps · number of trees per rep. Received for publication 1 Nov. 2016. Accepted for publication 5 Dec. 2016. This research was supported in part by grants from the Florida Citrus Research and Development Foundation. Mention of a trademark, warranty, proprietary product, or vendor does not imply an approval to the exclusion of other products or vendors that may also be suitable. The U.S. Department of Agriculture prohibits discrimination in all its programs and activities on the basis of race, color, national origin, gender, religion, age, disability, political beliefs, sexual orientation, and marital or family status. We acknowledge the technical assistance of Emily Domogtoy, James Salvatore and Wayne Brown. Corresponding author. E-mail: greg.mccollum@ ars.usda.gov. HORTSCIENCE VOL. 52(4) APRIL 2017 541 Poncirus trifoliata. Although previously not commonly used in Florida, currently there is considerable interest in ‘x639’ rootstock. In 2014, ‘x639’ was used for the production of 622,000 nursery trees, accounting for 14% of all nursery propagations and making it the second most widely propagated citrus rootstock in Florida (Kesinger, 2015). ‘Ray Ruby’ fruit are valued for their red flesh, low seed count, and pink blush that develop in the rind. In addition to their deep red color, ‘Ray Ruby’ juice is sweeter than it is for comparable varieties (Saunt, 1990). To be successful, new rootstock hybrids must produce not only good fruit yields but also fruit with acceptable quality. According to Castle (1995) and Castle et al. (2010), yield is the major factor when selecting citrus rootstocks, although rootstock effects on fruit and juice quality can impact financial returns, particularly for fresh fruit production (Castle, 2012; Castle et al., 2010). The objective of the work reported herein was to determine the effects of Table 2. Effects of rootstock on ‘Ray Ruby’ grapefruit quality parameters, 2011–12 harvest season.

Rootstock effects on metabolite composition in leaves and roots of young navel orange (Citrus sinensis L. Osbeck) and pummelo (C. grandis L. Osbeck) trees

Key messageRootstock variety influences leaf metabolic profiles in a grafted citrus tree, but influence also depends on the scion.AbstractProper selection of rootstocks in tree fruit crops such as citrus is important for successful production. Despite a large number of commercially available rootstocks, studies have mostly been limited to basic horticultural observations. We used untargeted gas chromatography-time of flight-mass spectrometry (GC-TOF MS) based metabolomics to understand the biochemical influence of rootstock on 2-year-old field-grown ‘Cara Cara’ navel orange (Citrus sinensis L. Osbeck) and ‘Hirado Buntan’ pummelo (C. grandis L. Osbeck) trees grown on four rootstock cultivars with different genetic background. Five hundred unique metabolites were quantified in all trees, of which 147 were chemically identified. In navel orange trees, 48 root metabolites differed significantly in concentrations among rootstocks, compared with 29 metabolites in pummelo trees. In navel orange trees, raffinose, conduritol beta-epoxide, allantoic acid, myo-inositol, gamma-tocopherol, and beta gentiobiose were among the compounds that contributed most to this variation. In pummelo trees, hexitol, allantoic acid, glucoheptulose, tryptophan, gamma-tocopherol, glycerol-3-galactoside, and raffinose were among the most discriminating metabolites, but only allantoic acid passed significance criteria. Rootstock was also found to influence the quantities of 226 metabolites in leaves of the navel orange scion. Conduritol-beta-epoxide and myo-inositol were among the metabolites most influenced by rootstock. In contrast, the influence of rootstock on the pummelo scion was less prominent, with only six metabolites displaying significant differences. Our findings demonstrate that rootstock variety can influence the metabolic profile of the leaves in a grafted tree, but that the extent of the effect is also influenced by the scion. The majority of root metabolites that discriminated most between rootstocks did not display the same rootstock-specific discrimination in the leaves, suggesting tissue specificity or limited movement across the graft union.

Marker assisted selection in citrus rootstock breeding based on a major gene locus 'Tyr1' controlling citrus nematode resistance.

Based on the former constructed ‘Tyr1’ locus genetic map in family 9145, from LB6-2 [Clementine mandarin (C. reticulata) × Hamlin orange (C. sinensis)] × Swingle citrumelo (C. paradise × P. trifoliata), 9 markers were chosen for application in evaluating their effectiveness in marker-assisted selection (MAS) for citrus rootstock breeding program from many F1 progeny of Poncirus trifoliata. As the mapping revealed that these markers were estimated within a range of 12.1 cM in the linkage group, and among them, SCO07 co-segregated with ‘Tyr1’, and 7A4R as the closest to ‘Tyr1’ with a distance of 1.5 cM, these markers were basically fitful to go MAS screening. The results of screening P. trifoliata F1 progeny indicated that all the markers were inherited in codominant fashion and most of them were heterozygous on PT (Pomery of P. trifoliata)., marker 4L17R/CfoI and 7A4(1407)/BfaI were proved to be consistently reliable for accurate scoring of genotypes and the revealed polymorphism was basically coincided with the citrus nematode resistant phenotype within tested populations. The polymorphic genotype with marker 4L17R/CfoI was found completely matched up with the phenotype of individuals that conferred high resistance to citrus nematode when the USDA hybrid rootstocks were screened. Utilization of these markers, especially the highly specific 4L17R/CfoI and 7A4(1407)/BfaI, should result in great benefit to world citrus industry for early selection in rootstock-breeding program.