Genome resequencing and transcriptome profiling reveal structural diversity and expression patterns of constitutive disease resistance genes in Huanglongbing-tolerant Poncirus trifoliata and its hybrids

Huanglongbing(HLB)is the most destructive bacterial disease of citrus worldwide.While most citrus varieties are susceptible to HLB,Poncirus trifoliata,a close relative of Citrus,and some of its hybrids with Citrus are tolerant to HLB.No specific HLB tolerance genes have been identified in P.trifoliata but recent studies have shown that constitutive disease resistance(CDR)genes were expressed at much higher levels in HLB-tolerant Poncirus hybrids and the expression of CDR genes was modulated by Candidatus Liberibacter asiaticus(CLas),the pathogen of HLB.The current study was undertaken to mine and characterize the CDR gene family in Citrus and Poncirus and to understand its association with HLB tolerance in Poncirus.We identified 17 CDR genes in two citrus genomes,deduced their structures,and investigated their phylogenetic relationships.We revealed that the expansion of the CDR family in Citrus seems to be due to segmental and tandem duplication events.Through genome resequencing and transcriptome sequencing,we identified eight CDR genes in the Poncirus genome(PtCDR1-PtCDR8).The number of SNPs was the highest in PtCDR2 and the lowest in PtCDR7.Most of the deletion and insertion events were observed in the UTR regions of Citrus and Poncirus CDR genes.PtCDR2 and PtCDR8 were in abundance in the leaf transcriptomes of two HLB-tolerant Poncirus genotypes and were also upregulated in HLB-tolerant,Poncirus hybrids as revealed by real-time PCR analysis.These two CDR genes seem to be good candidate genes for future studies of their role in citrus-CLas interactions.

Comparative transcriptome analysis during early fruit development between three seedy citrus genotypes and their seedless mutants

Identification of genes with differential transcript abundance(GDTA)in seedless mutants may enhance understanding of seedless citrus development.Transcriptome analysis was conducted at three time points during early fruit development(Phase 1)of three seedy citrus genotypes:Fallglo(Bower citrus hybrid(Citrus reticulata×C.reticulata×C.paradisi)×Temple(C.reticulata×C.sinensis)),grapefruit(C.paradisi),Pineapple sweet orange(C.sinensis),and their seedless mutants.Seed abortion in seedless mutants was observed at 26 days post anthesis(Time point 2).Affymetrix transcriptomic analysis revealed 359 to 1077 probe sets with differential transcript abundance in the comparison of seedless versus seedy fruits for each citrus genotypes and time points.The GDTA identified by 18 microarray probe sets were validated by qPCR.Hierarchical clustering analysis revealed a range of GDTA associated with development,hormone and protein metabolism,all of which may reflect genes associated with seedless fruit development.There were 14,9 and 12 genes found exhibiting similar abundance ratios in all three seedless versus seedy genotype comparisons at time point 1,2 and 3,respectively.Among those genes were genes coding for an aspartic protease and a cysteine protease,which may play important roles in seedless fruit development.New insights into seedless citrus fruit development may contribute to biotech approaches to create seedless cultivars.

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

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 14 weeks 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.