Small RNA Profiling Reveals Phosphorus Deficiency as a Contributing Factor in Symptom Expression for Citrus Huanglongbing Disease

HongweiZhao 0 RuobaiSun 0 UteAlbrecht 0 ChellappanPadmanabhan 0 AirongWang 0 Michael D.Coffey 0 ThomasGirke 0 ZonghuaWang 0 Timothy J.Close 0 MikealRoose 0 Raymond K.Yokomig 0 SvetlanaFolimonovah 0 GeorgiosVidalakis 0 RobertRousei 0 Kim D.Bowman 0 HailingJin 0 0 a Department of Plant Pathology and Microbiology, University of California , Riverside, CA 92521 , USA b Institute for Integrative Genome Biology, University of California , Riverside, CA 92521 , USA c Present address: Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University , Nanjing, 210095 , China d Department of Botany and Plant Sciences, University of California , Riverside, CA 92521 , USA e US Horticultural Research Laboratory, US Department of Agriculture, Agricultural Research Service , 2001 South Rock Road, Fort Pierce, FL 34945 , USA f Fujian Agriculture and Forestry University , Fuzhou, 350002 , China g San Joaquin Valley Agricultural Research Center, US Department of Agriculture, Agricultural Research Service , 9611 S. Riverbend Ave, Parlier, CA 93648 , USA h Citrus Research and Education Center, University of Florida , 700 Experiment Station Road, Lake Alfred, FL 33850 , USA i University of Florida , 2685 State Road 29 North, Immokalee, FL 34142 , USA Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the genus 'Candidatus Liberibacter' (Ca. L.). Powerful diagnostic tools and management strategies are desired to control HLB. Host small RNAs (sRNA) play a vital role in regulating host responses to pathogen infection and are used as early diagnostic markers for many human diseases, including cancers. To determine whether citrus sRNAs regulate host responses to HLB, sRNAs were profiled from Citrus sinensis 10 and 14weeks post grafting with Ca. L.asiaticus (Las)-positive or healthy tissue. Ten new microRNAs (miRNAs), 76 conserved miRNAs, and many small interfering RNAs (siRNAs) were discovered. Several miRNAs and siRNAs were highly induced by Las infection, and can be potentially developed into early diagnosis markers of HLB.miR399, which is induced by phosphorus starvation in other plant species, was induced specifically by infection of Las but not Spiroplasma citri that causes citrus stubborn-a disease with symptoms similar to HLB. We found a 35% reduction of phosphorus in Las-positive citrus trees compared to healthy trees. Applying phosphorus oxyanion solutions to HLB-positive sweet orange trees reduced HLB symptom severity and significantly improved fruit production during a 3-year field trial in south-west Florida. Our molecular, physiological, and field data suggest that phosphorus deficiency is linked to HLB disease symptomology. - INTRODUCTION Huanglongbing (HLB), also called citrus greening, is one of the most destructive citrus diseases threatening the global citrus industry. HLB was first reported in Asia about a century ago, then in South Africa and South America (Gottwald, 2010). In the Americas, HLB was discovered in Sao Paulo, Brazil, in 2004, and subsequently in Florida and Texas, and more recently in California in 2012 (Stokstad, 2012). HLB is associated with phloem-restricted Gram-negative bacteria that belong to the genus Candidatus Liberibacter (Ca. L.) and are vectored by insects of the Psyllidae family (Bove, 2006). At least three members of the Ca. L. genus have been associated with HLB: Ca. L. africanus (Laf), Ca. L. americanus (Lam), and Ca. L. asiaticus (Las). In the US alone, annual HLB managementrelated expenses have totaled about 1.2 billion dollars (Perez etal., 2011), and it has been estimated that HLB management has increased citrus production costs by 4050% in affected areas (Stokstad, 2012). Current strategies for HLB management consist of control of the psyllid vectors, identification and removal of infected trees, and the use of pathogen-tested citrus nursery stocks for replants and new orchards. 1 To whom correspondence should be addressed. E-mail , tel. 9518277995, fax 9518274294. 2 These authors contributed equally to this work. The Author 2013. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/sst002, Advance Access publication 5 January 2013 Received 17 October 2012; accepted 21 December 2012 Although advances have been made in sensitive HLB diagnostics (Lin etal., 2010), the current polymerase chain reaction (PCR)-based techniques are dependent on detection of the DNA of the presumed causal bacteria and are reliable when bacterial cells or DNA is present in the tested sample. In some cases, disease symptoms are not present. This can be problematic sometimes, since the titer and distribution of the HLB-associated bacteria within citrus trees, types of tissue infected, and degree of disease progression are variable, especially in recently infected trees (Bove, 2006; Li etal., 2007; Tatineni et al., 2008; Li et al., 2009; Folimonova and Achor, 2010). Thus, it is important to develop new diagnostic markers that can detect infection independent of pathogen presence in a tested sample and prior to symptom development. Symptoms of HLB include blotchy mottled leaves, sections of yellow and underdeveloped vegetative growth, premature fruit drop, and in some cases small, off-flavored fruit with aborted seeds. The mechanisms of HLB pathogenesis and host responses are largely unknown. In the citrus relative Poncirus trifoliata and some of its hybrids that are used as rootstocks, tolerance has been observed (Folimonova et al., 2009; Albrecht and Bowman, 2011), but no HLB-resistant citrus scion cultivars have been identified. Many genes are differentially expressed between healthy and Las-positive plants (Albrecht and Bowman, 2008, 2012), manifesting the hosts effort against the bacterial infection. Previous studies in systems such as Arabidopsis have demonstrated that some host small RNAs (sRNAs) are rapidly and specifically induced by pathogen infection and contribute to the gene expression reprogramming in host defense responses (Katiyar-Agarwal etal., 2006; Navarro etal., 2006; Katiyar-Agarwal etal., 2007; Wang etal., 2011; Zhang etal., 2011b). Similarly, specific panels of human miRNAs have been used for early diagnosis of many human diseases, including various cancers (Iorio and Croce, 2009; Fabbri, 2010; Ferracin etal., 2010). In this study, we profiled sRNAs from both Laspositive and healthy sweet orange (Citrus sinensis (L.) Osbeck) plants and identified a panel of microRNAs (miRNAs) and small interfering RNAs (siRNAs) that were highly induced by Las infection. These sRNAs can potentially be developed into early diagnosis markers. Induction of miR399 in Las-positive trees corresponds to our finding that Las-positive plants had severe phosphorus (P) deficiency. MiR399 induction is associated with phosphorus deficiency in Arabidopsis and other species (Fujii etal., 2005; Bari etal (...truncated)