Discovery,Functional Analysis And Disease Resistance Utilization Of Upland Cotton Lesion Mimic Mutant

Programmed cell death(PCD)is a normal process in plant development and one type of responses when normal development is perturbed or under environmental stresses.Hypersensitive response(HR)which results in cell death at the sites of infection by pathogen is a form of PCD associated with disease resistance.Some lesion mimic mutants(LMM)show HR-like cell death on leaves and enhanced immunity than their corresponding wild type plants.So,LMMs are elite material to study the PCD and its connection to defense signaling pathways in plants.Fungal disease is a devastating threat to both global food security and crop yields.Verticillium-wilt is a worldwide fungal disease eausing severe problems to most economically important crops,such as potatoes,tomatoes and cotton.Cotton(Gossypium spp.)is an important economic crop and the largest source of textile fiber in the world.Upland cotton is one of the main cultivated cotton species planted in China accounting for more than 90%of the cotton production.However,cultivated cotton varieties of high reistance to Verticillium wilt,are rare,mainly because of lack of Upland cotton germplasms which are nature immune or highly resistant to Verticillium wilt.We have identified a lesion mimic mutant named Ghlmm in Upland cotton.As we know,it is t:he first one of this type in the mutation found in cotton.The F1 and F2 mapping populations were developed by a cross between Ghlmm and TM-1,a genetic standard line of G.hirsutum.The phenotypic assays showed that all Fi plants exhibit the wild-type phenotype and the segregation of the F2 plants meets the Mendelian ratio of 3:1(554 individuals with wild type phenotype and 209 individuals with lesion mimic phenotype,x2=2.202<x20.05,1=3.84),suggesting that the lesion mimic phenotype was controlled by a single nuclear recessive gene.A linkage analysis with 763 F2 plants delimited the target gene to a 371-kb DNA segment by markers NAU7928and S2393 on Chr.D5,we therefore named the target gene as GhLMMD.In this region 25 DNA intervals with high probability of containing exons were predicted.The transcriptome analysis in TM-1 revealed that,there are 12 genes whose FPKM value was above 1.0.It was found that among them,two genes,named Gh_D05G2237 and Gh_D05G2254,were significantly decreased in transcript abundance in Ghlmm compared with in WO.In order to identify GhLMMD.the DNA and cDNA of Gh_D05G2237 and Gh_D05G2254 in Ghlmm,WO,and TM-1 respectively were amplified and sequenced.Sequence comparisons showed that no nucleotide differences in Gh_D05G2254 were detected between Ghlmm and WO.TM-1.However,a point mutation(G-to-T transition at 127th position within the coding region)was detected in Gh_D05G2237 of Ghlmm compared with that of WO and TM-1,which resulted in a pre-termination(GGA to TGA)in the protein.The suppression of Gh_D05G2237.but not Gh_D05G2254,led to a lesion mimic phenotype similar to that in Ghlmm.Based on the point mutation single nucleotide polymorphism(SNP)primers which could specifically detect the 127th-T genotype but not the 127th-G were developed,and were successfully used with the genotyping 209 F2 plants exhibiting the Ghlmm phenotype.The BLAST analysis showed that GhLMMD encodes 5-aminolevulinic acid dehydratase(ALAD).In plants,ALAD is an enzyme catalyzing the porphobilinogen(PBG)formation by condensing two molecules of the 5-aminolevulinic acid(ALA).GhLMMD and its corresponding gene in the A subgenome,GhLMMA show similar expression patterns in all tested cotton tissues,with higher expression levels in leaves,early stage ovules and developing fibers.The subcellular localization showed that both GhLMMA and GhLMMD proteins localize in chloroplasts.The phylogenetic analysis revealed that no more than two ALAD genes was found in each tested species and the ALAD motifs share high sequence similarities,indicating that ALAD is an ancient and conserved protein in plants.Transcript abundance of GhLMMD and GhLMMA were respectively monitored by qRT-PCR using gene-specific primers.It was showed that the transcript abundance of GhLMMD was indeed decreased in Ghlmm compared with WO,whereas,the GhLMMA expression was not altered in both Ghlmm and WO.The GhLMMD mutation reduce the ALAD enzyme activity and result in the over-accumulation of ALA and generate reactive oxygen species(ROS).Infection assays showed that Ghlmm confer cotton resistance to Verticillium dahliae infection.In order to obtain comprehensive insights into the molecular basis for the increased resistance,an RNA-seq was performed in Ghlmm and WO.The data revealed that a total of 2069 differential expression genes(q<0.05 and a fold change>2),with 1541 upregulated and 528 downregulated genes,were detected in Ghlmm compared with WO.The GO analysis indicating the immunity-related pathway initiated in Ghlmm.The SA levels have significantly increased in the stems and leaves of Ghlmm compared to WO.The SA signaling marker genes,such as the PR genes and NPRI were significantly upregulated in Ghlmm,particularly in the leaves and stems.The expression of EDSlI PAD4 and PAL was induced by the H2O2 treatment in the leaves of WO.Furthermore,their expression was much higher in Ghlmm than in WO.The enzyme activities of CAT,reported to be inhibited by the SA,were drastically decreased in Ghlmm.Blocking SA biosynthesis pathway by application with AIP(chemical of 2-aminoindan-2-phosphonic acid),a highly specific inhibitor of PAL,significantly decreased levels of SA,ROS in both Ghlmm and WO and eliminate the necrotic lesions in leaves of Ghlmm.GhLMM gene may have dosage effect in regulating the degree of PCD,defense responses and resistance to the V.dahliae infection.We first started the experiments by regulating the endogenous enzyme activities of the ALAD of cotton leaves by spraying different concentrations of the LA solutions.The results showed that a 20 mM LA treatment could cause an evident lesion mimic phenotype,while a 0-10 mM LA treatnent did not lead to visible leaf lesions.The increases in the PR gene expression and levels of ALA,H2O2 and SA were parallel correlated to the increased concentrations of the LA applied during treatment.We next explored the effects of the GhLMM gene dosage on disease resistance.The resistance levels of WO?Ghlmm and Fi to the V.dahliae infection were as follows:mutant>F1>wild type.The enzyme activity of the ALAD,CAT and the contents of ALA,PBG and H2O2,SA in the F1 plants were also at mid-level between the parents.Similarly,the PR gene expression in these lines exhibited a gene dosage-dependent pattem.(l)Four copies of GhLMM maintain the plants in good growth conditions without the ALA accumulation and defense activation;(2)Three copies exhibit no lesion on the leaves but lead to an increased level of ALA,ROS production and disease resistance.(3)Two copies display a significantly increased disease resistance level,but the lesion mimic phenotype occurs due to the ALA over-accumulation.(4)A simultaneous suppression of the GhLMM homeologs in tetraploid cotton bloeks the tetrapyrrol biosynthesis pathway and eventually leads to plant death.